Assay systems for genetic analysis

ABSTRACT

The present invention provides assay systems and methods for detection of copy number variation at one or more loci and polymorphism detection at one or more loci in a mixed sample from an individual.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/205,490, filed Aug. 8, 2011, which is a continuation-in-part of U.S.application Ser. No. 13/013,732, filed Jan. 25, 2011, which claimspriority to U.S. Ser. No. 61/371,605, filed Aug. 6, 2010, which areherein incorporated by reference in their entities.

INCORPORATION-BY-REFERENCE OF A SEQUENCE LISTING

The content of the following submission of ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CFR) of the Sequence Listing (file name:50876-502C03US_SEQLIST.txt, date recorded: Nov. 28, 2016, size: 2,995bytes).

FIELD OF THE INVENTION

This invention relates to the detection and quantification of geneticvariation in mixed samples in a single assay.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Genetic abnormalities account for a wide number of pathologies,including pathologies caused by chromosomal aneuploidy (e.g., Downsyndrome), germline mutations in specific genes (e.g., sickle cellanemia), and pathologies caused by somatic mutations (e.g., cancer).Diagnostic methods for determining such genetic anomalies have becomestandard techniques for identifying specific diseases and disorders, aswell as providing valuable information on disease source and treatmentoptions.

Although conventional technology provides detection methods for thesedifferent genetic abnormalities, it currently requires differenttechniques to interrogate different classes of mutations. An assayproviding detection of a copy number variation with simultaneousdetection of individual gene polymorphisms would be a powerful tool inthe treatment. The present invention addresses this need.

There is thus a need for non-invasive methods of screening for geneticabnormalities, including copy number variations, in mixed samplescomprising normal and putative abnormal DNA. The present inventionaddresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention provides assay systems and related methods fordetermining genetic variation in mixed samples comprising genomicmaterial (e.g., cell free DNA) from a major and one or more minorsources from a subject. More particularly, the present inventionprovides assay systems and methods for detection of copy numbervariation of one or more loci and detection of polymorphisms in one ormore loci in a sample comprising nucleic acids from a major source and aminor source within an individual. The present invention utilizes asingle assay system that allows the identification of both genetic copynumber variations (CNVs) and single gene polymorphisms, and the abilityto distinguish between these CNVs and polymorphisms in the major andminor sources.

In one aspect, the assay system utilizes amplification and detection ofselected loci in a mixed sample from an individual to identify the copynumber and presence of polymorphisms in selected loci from a major andone or more minor sources.

In one specific aspect, the invention provides single assay systems withthe ability to determine 1) the presence or absence of CNVs at one ormore loci and; 2) the presence or absence of polymorphisms at one ormore loci in a mixed sample from an individual. The assay system canspecifically detect copy number of selected loci and polymorphismspresent in selected loci from a minor source within the mixed sample,and to distinguish this from the copy number and polymorphisms presentin selected loci from a major source in the mixed sample. Preferably,the loci are analyzed through use of cell free nucleic acids, andpreferably the cell free nucleic acids analyzed in the assay system arecell free DNA (cfDNA).

Thus, in a specific aspect, the invention provides an assay system fordetection of the presence or absence of a copy number variation (CNV) ofa genomic region and presence or absence of one or more polymorphisms ina mixed sample using a single assay, the assay comprising the steps ofintroducing a first set of fixed sequence oligonucleotides to a mixedsample under conditions that allow the fixed sequence oligonucleotidesto specifically hybridize to complementary regions on one or more lociin or associated with a genomic region; introducing a second set offixed sequence oligonucleotides comprising universal primer regions tothe mixed sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions onone or more loci with a putative polymorphism; ligating the hybridizedoligonucleotides to create contiguous ligation products complementary tothe loci corresponding to a genomic region and/or the loci with aputative polymorphism; amplifying the contiguous ligation products tocreate amplification products; and detecting the amplification products.The detection of the amplification products correlates to the copynumber of one or more genomic regions and the presence or absence of apolymorphism in one or more loci in the mixed sample.

In certain aspects, the fixed sequence oligonucleotides hybridize toadjacent regions in the loci, and the fixed sequence oligonucleotidesare directly ligated during the ligation step of the assay. In otheraspects, the fixed sequence oligonucleotides hybridize to non-adjacentregions in the loci that are separated by one, a few, or severalnucleotides, and the region between the fixed sequence oligonucleotidesis used as a template for primer extension to close the gap between thefixed sequence oligonucleotides prior to the ligation step.

Thus, in a second aspect, the invention provides an assay system fordetection of the presence or absence of CNV of a genomic region andpresence or absence of one or more polymorphisms in a mixed sample usinga single assay, the assay comprising the steps of: introducing a firstset of fixed sequence oligonucleotides to a mixed sample underconditions that allow the fixed sequence oligonucleotides tospecifically hybridize to complementary regions on one or more loci inor associated with a genomic region; introducing a second set of fixedsequence oligonucleotides to the mixed sample under conditions thatallow the fixed sequence oligonucleotides to specifically hybridize tocomplementary regions on one or more loci with a putative polymorphism;extending the region between the first and second hybridizedoligonucleotide of at least one fixed sequence oligonucleotide set witha polymerase and dNTPs to create two adjacently hybridized fixedsequence oligonucleotides from that set; ligating the hybridizedoligonucleotides to create contiguous ligation products complementary tothe loci corresponding to a genomic region and/or the loci with aputative polymorphism; amplifying the contiguous ligation products usingthe universal primer regions to create amplification products; anddetecting the amplification products. The detection of the amplificationproducts correlates to the copy number of one or more genomic region andthe presence or absence of a polymorphism in one or more loci in themixed sample.

In a preferred aspect, the fixed sequence oligonucleotides used tointerrogate the specific loci comprise universal primer regions that arecommon to substantially all of the fixed sequence oligonucleotides usedin a particular assay. This allows substantially all of the ligationproducts produced in a single assay to be amplified, isolated and/oranalyzed using similar mechanisms (e.g., sequence determination orhybridization). Use of such universal primer regions obviates the needfor individually distinguishable detectable moieties associated withparticular loci or alleles, and provides a more efficient andcost-effective mechanisms for multiplexing interrogation and/or analysisof multiple loci from one or multiple samples. In a preferred aspect,the universal primer regions are used in sequence determination of theamplification products. In another preferred aspect, the same universalprimer regions are used in the fixed sequence oligonucleotides used fordetection of genomic regions and the fixed sequence oligonucleotidesused for detection of polymorphisms.

Each set of fixed sequence nucleic acids is designed to hybridize to atleast two separate regions in a selected locus. In preferred aspects,two or more separate oligos are used in a set to hybridize to theseregions to provide adjacent nucleic acids complementary to the selectedloci. In some aspects, however, a set can comprise a single probe withtwo or more distinct non-adjacent regions that are complementary to theselected loci (e.g., padlock probes), as described in more detailherein. The sets of fixed sequence oligos can be provided in the assaysequentially or simultaneously in the assay.

The genomic region can be a single locus, or it may be a larger region,up to and including a chromosome. For determination of larger genomicregions, sets of loci may be used to determine the size and in certainaspects the boundaries of such genomic regions for which CNV has beendetected.

In certain aspects, the amplification products of the assay are isolatedprior to detection. Preferably, the amplification products are isolatedas individual molecules prior to detection. These isolated amplificationproducts can optionally be further amplified to create identical copiesof all or a portion of the individual amplification products prior todetection, or further amplified to create identical copies of moleculescomplementary to all or a portion of the individual amplificationproducts prior to detection.

The multiplexed assays of the invention allow the analysis of 5 or more,preferably 10 or more, preferably 16 or more, preferably 20 or more,preferably 30 or more, preferably 32 or more, preferably 40 or more,preferably 48 or more, preferably 50 or more, preferably 60 or more,preferably 70 or more, preferably 80 or more, preferably 90 or more, andmore preferably 96 or more selected loci simultaneously. These selectedloci may be different loci from a single sample, or they may be locifrom two or more individuals. In the latter case, at least one of thetwo fixed sequence oligonucleotides used for analysis of a selectedlocus may comprise a sample identifier (e.g., a “sample index”) thatwill allow the locus to be associated with a particular sample.Alternatively, a sample index may be added during amplification of theligation product by using a primer comprising the sample index.

Preferably, at least one locus interrogated for CNV in a mixed sample isdifferent from all loci interrogated for polymorphisms in the mixedsample. In specific aspects, several loci interrogated for CNV in amixed sample are different from the loci interrogated for polymorphismsin the mixed sample. In more specific aspects, the majority of lociinterrogated for CNV in a mixed sample are different from the lociinterrogated for polymorphisms in the mixed sample.

In some aspects of the invention, the fixed sequence oligonucleotideshybridize to adjacent regions on a locus, and ligation of theseoligonucleotides results in a ligation product that joins the two fixedsequence oligonucleotides. In other aspects, the fixed sequenceoligonucleotides hybridize to non-adjacent regions on a locus, and theregions between the fixed sequence oligonucleotides is extended using apolymerase and dNTPs. In preferred aspects, the fixed sequenceoligonucleotides hybridize to non-adjacent regions on a locus, and oneor more bridging oligonucleotides hybridize to the region between andadjacent to the set of fixed sequence oligonucleotides. In certainpreferred aspects, the bridging oligonucleotides used can providecontent information on polymorphisms in the loci as well as informationon frequency of loci for CNV analysis.

In other preferred aspects, the interrogation of these loci utilizesuniversal amplification techniques that allow amplification of multipleloci in a single amplification reaction. The selected nucleic acids fordetection of both the CNV and polymorphisms using the assay system ofthe invention can be amplified using universal amplification methodsfollowing the initial selective amplification from the mixed sample. Theuse of universal amplification allows multiple nucleic acids regionsfrom a single or multiple samples to be amplified using a single orlimited number of amplification primers, and is especially useful inamplifying multiple selected regions in a single reaction.

Thus, in a preferred aspect of the invention, sequences complementary toprimers for use in universal amplification are introduced to theselected loci during or following selective amplification. Preferablysuch sequences are introduced to the ends of such selected nucleicacids, although they may be introduced in any location that allowsidentification of the amplification product from the universalamplification procedure.

In certain preferred aspects, one or both of the primers used comprise asample index or other identifier. In a specific aspect, a sample indexis included in one or more of the universal primers. The sample index isincorporated into the amplification products, and amplification productsfrom different samples may then be combined. The sample index ispreferably detected concurrently with the detection of the CNV orchromosomal abnormality and the detection of polymorphism such that theCNV and polymorphism may be properly assigned to the sample of origin.

Frequencies of selected loci can be determined for a genomic region ofinterest and compared to the frequencies of loci of one or more othergenomic regions of interest and/or one or more reference genomic regionsto detect potential CNVs based on loci frequencies in the mixed sample.

In the assay systems of the invention, the amplification products areoptionally isolated prior to detection. When isolated, they arepreferably isolated as individual molecules to assist in subsequentdetection. Following isolation, the amplification products can befurther amplified to create identical copies of all or a portion of theindividual amplification products prior to detection. Alternatively, theisolated amplification products can be further amplified to createidentical copies of molecules complementary to all or a portion of theindividual amplification products prior to detection.

Various methods of detection of CNVs can be employed in conjunction withthe detection of the polymorphisms in the assay systems of theinvention. In one general aspect, the assay system employs a method fordetermination of a CNV in one or more loci in a mixed sample, comprisingthe steps of amplifying one or more selected nucleic acids from a firstgenomic region of interest in a mixed sample; amplifying one or moreselected nucleic acids from a second locus of interest in the mixedsample, determining the relative frequency of the selected loci,comparing the relative frequency of the selected loci, and identifyingthe presence or absence of a CNV based on the compared relativefrequencies of the selected nucleic acids from the first and secondloci. Preferably, the assay method amplifies two or more selected locifrom different genomic regions, although the loci may be located in thesame general genomic region for confirmation of CNVs arising fromchromosomal abnormalities rather than CNVs from a single locus.

In other aspects, the bridging oligos are degenerate oligos. In otheraspects, the bridging oligos are provided as pools of oligos with randomsequences that comprise substantially every combination for theparticular size of the bridging oligo used with the fixed sequenceoligonucleotides.

Although the aspects of the invention using bridging oligonucleotidesare described primarily using a single bridging oligo, it is envisionedthat multiple bridging oligos that hybridize to adjacent, complementaryregions between fixed sequence oligonucleotides can be used in thedescribed methods.

In one preferred aspect, the invention provides an assay system fordetection of the presence or absence of copy number variation (CNV) of agenomic region and presence or absence of one or more polymorphisms in amixed sample using a single assay, the assay comprising the steps of:introducing a first set of fixed sequence oligonucleotides comprisinguniversal primer regions to a mixed sample under conditions that allowthe fixed sequence oligonucleotides to specifically hybridize tocomplementary regions on one or more loci in or associated with agenomic region; introducing a second set of fixed sequenceoligonucleotides comprising universal primer regions to the mixed sampleunder conditions that allow the fixed sequence oligonucleotides tospecifically hybridize to complementary, non-adjacent regions on one ormore loci with a putative polymorphism; introducing one or more bridgingoligonucleotides under conditions that allow the bridgingoligonucleotides to specifically hybridize to regions in the locibetween the regions complementary to the fixed sequence oligonucleotidesof the sets; ligating the hybridized oligonucleotides to createcontiguous ligation products complementary to the loci corresponding toa genomic region and/or the loci with a putative polymorphism;amplifying the contiguous ligation products using the universal primerregions to create amplification products; and detecting theamplification products. The detection of the amplification productscorrelates to the copy number of one or more genomic region and thepresence or absence of a polymorphism in one or more loci in the mixedsample.

In another specific aspect, the invention provides an assay system fordetection of the presence or absence of CNV of a genomic region andpresence or absence of one or more polymorphisms in a mixed sample usinga single assay, the assay comprising the steps of: introducing a firstset of fixed sequence oligonucleotides to a mixed sample underconditions that allow the fixed sequence oligonucleotides tospecifically hybridize to complementary regions on one or more loci inor associated with a genomic region; introducing a second set of fixedsequence oligonucleotides to the mixed sample under conditions thatallow the fixed sequence oligonucleotides to specifically hybridize tocomplementary regions on one or more loci with a putative polymorphism;introducing one or more bridging oligonucleotides under conditions thatallow the bridging oligonucleotides to specifically hybridize to regionsin the loci between the regions complementary to the fixed sequenceoligonucleotides of the set; extending the region between at least onefixed sequence oligonucleotide and a bridging oligonucleotide with apolymerase and dNTPs to create adjacently hybridized fixed sequenceoligonucleotides and bridging oligonucleotides; ligating the hybridizedoligonucleotides to create contiguous ligation products complementary tothe loci corresponding to a genomic region and/or the loci with aputative polymorphism; amplifying the contiguous ligation products usingthe universal primer regions to create amplification products; anddetecting the amplification products. The detection of the amplificationproducts correlates to the copy number of one or more genomic region andthe presence or absence of a polymorphism in one or more loci in themixed sample.

In preferred aspects of the aspects of the invention using bridgingoligonucleotides, the first and second set of fixed sequenceoligonucleotides are introduced prior to introduction of the bridgingoligonucleotides. More preferably, the unhybridized fixed sequenceoligonucleotides are removed prior to introduction of the bridgingoligonucleotides. In some aspects, the bridging oligonucleotides areintroduced simultaneously with the ligation mixture. In other aspects,the hybridization products of the fixed sequence oligonucleotides andthe locus are isolated prior to introduction of the bridgingoligonucleotides.

In a preferred aspect, the assay system provides highly multiplexed lociinterrogation using one or more common bridging oligonucleotides thatare complementary to regions in two or more interrogated loci. Thus, thenumber of bridging oligonucleotides used in the multiplexed assay systemwill be less than the number of loci interrogated in the assay. Incertain specific aspects, the assay system uses a pool of bridgingoligonucleotides that are each designed to be compatible with two ormore loci interrogated using the assay system of the invention. In theseaspects, the bridging oligonucleotides used in the multiplexed assay arepreferably designed to have a T_(m) in a range of ±5° C., morepreferably in a range of ±2° C.

In certain aspects, the assay system multiplexes loci interrogationusing one or more common bridging oligonucleotides that arecomplementary to regions in two or more interrogated loci. Thus, thenumber of bridging oligonucleotides used in the multiplexed assay systemwill be less than the number of loci interrogated in the assay. Incertain specific aspects, the assay system uses a pool of bridgingoligonucleotides that are each designed to be compatible with two ormore loci interrogated using the assay system of the invention.

In certain aspects, the bridging oligonucleotides are between 2-45nucleotides in length. In a specific aspect, the bridgingoligonucleotides are between 3-9 nucleotides in length. In yet anotherspecific aspect, the oligonucleotides are between 10-30 nucleotides inlength.

The loci interrogated for CNV can in some instances be indicative of aduplication of a larger genomic region, e.g., all or part of achromosome. Preferably, the assay systems can distinguish the copynumber of these loci between a major source and a minor source within amixed sample.

In such aspects, the invention provides assay systems that comprise asingle assay system with the ability to detect within a mixed samplefrom an individual 1) presence or absence of a chromosomal abnormalityassociated with one or more CNV; and 2) presence or absence of one ormore polymorphisms at one or more selected locus. The presence orabsence of the chromosomal abnormality is preferably detected throughthe use of informative loci that allow the assay system to distinguishbetween nucleic acids from a major source and a minor source.

Thus, in a specific aspect, the invention provides an assay system fordetection of the presence or absence of a chromosomal abnormality andthe presence or absence of one or more polymorphisms in a mixed sampleusing a single assay, the assay comprising the steps of: introducing afirst set of fixed sequence oligonucleotides comprising universal primerregions to a mixed sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions onone or more loci with a putative polymorphism; introducing a second setof fixed sequence oligonucleotides comprising universal primer regionsto the mixed sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions ontwo or more loci from a first chromosome; introducing a third set offixed sequence oligonucleotides comprising universal primer regions tothe mixed sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions ontwo or more loci from a second chromosome; introducing one or morebridging oligonucleotides under conditions that allow the bridgingoligonucleotides to specifically hybridize to regions in the locibetween the regions complementary to the fixed sequence oligonucleotidesof the set; ligating the hybridized oligonucleotides to createcontiguous ligation products complementary to the nucleic acids;amplifying the contiguous ligation products to create amplificationproducts; and detecting the amplification products. The detection of theamplification products correlates to the presence or absence of achromosomal abnormality and the presence or absence of a polymorphism inone or more loci in the mixed sample.

In another specific aspect, the invention provides an assay system fordetection of the presence or absence of a chromosomal abnormality andpresence or absence of one or more polymorphisms in a mixed sample usinga single assay, the assay comprising the steps of: introducing a firstset of fixed sequence oligonucleotides comprising universal primerregions to a mixed sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions onone or more loci with a putative polymorphism; introducing a second setof fixed sequence oligonucleotides comprising universal primer regionsto the mixed sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions ontwo or more loci from a first chromosome; introducing a third set offixed sequence oligonucleotides comprising universal primer regions tothe mixed sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions ontwo or more loci from a second chromosome; introducing one or morebridging oligonucleotides under conditions that allow the bridgingoligonucleotides to specifically hybridize to regions in the locibetween the regions complementary to the fixed sequence oligonucleotidesof the set; extending the region between the first and second hybridizedoligonucleotide of at least one fixed sequence oligonucleotide set witha polymerase and dNTPs to create two adjacently hybridized fixedsequence oligonucleotides from that set; ligating the hybridizedoligonucleotides to create contiguous ligation products complementary tothe nucleic acids; amplifying the contiguous ligation products to createamplification products; and detecting the amplification products. Thedetection of the amplification products correlates to the presence orabsence of a chromosomal abnormality and the presence or absence of apolymorphism in one or more loci in the mixed sample.

In some instances, the chromosomal abnormality is associated with geneduplication or loci expansion on a chromosome of interest. In otherinstances, the chromosomal abnormality is associated with atranslocation resulting in the presence of an extra portion of achromosome in the genome. In yet other instances, the chromosomalabnormality is associated with aneuploidy of a chromosome of interest.

Thus, in one aspect, the invention provides an assay system fordetection of the presence or absence of a chromosomal abnormality andthe presence or absence of polymorphisms at one or more loci using asingle assay, the assay comprising the steps of introducing a first setof fixed sequence oligonucleotides to a mixed sample under conditionsthat allow the fixed sequence oligonucleotides to specifically hybridizeto complementary regions in two or more nucleic acids corresponding toinformative loci on two or more chromosomes; introducing a second set offixed sequence oligonucleotides to the mixed sample under conditionsthat allow the fixed sequence oligonucleotides to specifically hybridizeto complementary regions in nucleic acids indicative of the presence orabsence of polymorphisms at one or more loci; introducing one or morebridging oligonucleotides under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions inthe nucleic acids, wherein one or more bridging oligonucleotides arecomplementary to a region of the nucleic acids between and immediatelyadjacent to the regions complementary to the fixed sequenceoligonucleotides of each set; ligating the hybridized oligonucleotidesto create contiguous ligation products complementary to the nucleicacids; amplifying the contiguous ligation products to createamplification products; and detecting the amplification products.Detection of the amplification product correlates to the detection ofthe loci in the mixed sample, and can be used to determine the quantityof loci associated with the presence or absence of the chromosomalabnormality and the genetic status of one or more loci in the mixedsample. Detected levels of these nucleic acids can thus be used todetermine the presence or absence of the chromosomal abnormality and thepresence or absence of a polymorphism at one or more loci from a majorsource and/or a minor source within a mixed sample.

Thus, in one aspect, the invention provides an assay system fordetection of the presence or absence of a chromosomal abnormality andthe presence or absence of polymorphisms at one or more loci using asingle assay, the assay comprising the steps of introducing a first setof fixed sequence oligonucleotides to a mixed sample under conditionsthat allow the fixed sequence oligonucleotides to specifically hybridizeto complementary regions in two or more nucleic acids corresponding toinformative loci on two or more chromosomes; introducing a second set offixed sequence oligonucleotides to the mixed sample under conditionsthat allow the fixed sequence oligonucleotides to specifically hybridizeto complementary regions in nucleic acids indicative of the presence orabsence of polymorphisms at one or more loci; introducing one or morebridging oligonucleotides under conditions that allow the bridgingsequence oligonucleotides to specifically hybridize to complementaryregions in the nucleic acids, wherein one or more bridgingoligonucleotides are complementary to a region of the loci between theregions complementary to the fixed sequence oligonucleotides of each setand immediately adjacent to a first fixed sequence oligonucleotides;extending the region between the bridging oligonucleotide and the secondfixed sequence oligonucleotide using polymerase and dNTPs; ligating thehybridized oligonucleotides to create contiguous ligation productscomplementary to the nucleic acids; amplifying the contiguous ligationproducts to create amplification products; and detecting theamplification products. Detection of the amplification productcorrelates to the detection of the loci in the mixed sample, and can beused to determine the quantity of loci associated with the presence orabsence of the chromosomal abnormality and the genetic status of one ormore loci in the mixed sample. Detected levels of these nucleic acidscan thus be used to determine the presence or absence of the chromosomalabnormality and the presence or absence of a polymorphism at one or moreloci from a major source and/or a minor source within a mixed sample.

In another aspect, the present invention utilizes techniques that allowthe identification of both CNVs and infectious agents in a mixed sample.This may be especially helpful to monitor patients in which the clinicaloutcome may be compromised by the presence of an infectious agent. Forexample, a patient that has undergone a transplant will likely be takingimmunosuppressant medication, and so more prone to infection in general.Similarly, pregnant women have changes in their immune system and thusmay be more susceptible to infection with pathogens that may have anadverse effect on the mother and/or fetus. Also, certain types of cancerare associated with infectious agents (e.g., liver cancer associatedwith hepatitis B and C infections, cervical cancer associated with humanpapilloma virus infection), and identification of the infectious agentsmay be informative in predicting clinical outcome or determining thepreferred course of medical treatment for the patient.

Thus, in certain aspects, the invention provides an assay system fordetection of the presence or absence of genetic copy number variation(CNV) of a genomic region and the presence or absence of an infectiousagent in a mixed sample using a single assay, the assay comprising thesteps of: introducing a first set of fixed sequence oligonucleotides toa mixed sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions onone or more loci in or associated with a genomic region; introducing asecond set of fixed sequence oligonucleotides to the mixed sample underconditions that allow the fixed sequence oligonucleotides tospecifically hybridize to complementary regions in nucleic acidsindicative of an infectious agent; ligating the hybridizedoligonucleotides to create a contiguous ligation product complementaryto the nucleic acids; amplifying the contiguous ligation product tocreate amplification products; and detecting the amplification products.The detection of the amplification products correlates to copy number ofthe genomic region and the presence or absence of an infectious agent inthe mixed sample.

In other certain aspects. The invention provides an assay system fordetection of the presence or absence of copy number variation (CNV) of agenomic region, the presence or absence of one or more polymorphisms,and the presence or absence of an infectious agent in a mixed samplefrom an individual using a single assay, the assay comprising the stepsof: introducing a first set of fixed sequence oligonucleotides to amixed sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions onone or more loci in or associated with a genomic region; introducing asecond set of fixed sequence oligonucleotides to the mixed sample underconditions that allow the fixed sequence oligonucleotides tospecifically hybridize to complementary regions on one or more loci witha putative polymorphism; introducing a third set of fixed sequenceoligonucleotides to the mixed sample under conditions that allow thefixed sequence oligonucleotides to specifically hybridize tocomplementary regions in nucleic acids indicative of an infectiousagent; ligating the hybridized oligonucleotides to create a contiguousligation product complementary to the nucleic acids; amplifying thecontiguous ligation product to create amplification products; anddetecting the amplification products. The detection of the amplificationproducts correlates to copy number of the genomic region, the presenceor absence of a polymorphism at one or more loci, and the presence orabsence of an infectious agent in the mixed sample.

In these aspects, the assays can also comprise introducing one or morebridging oligonucleotides under conditions that allow the bridgingoligonucleotides to specifically hybridize to complementary regions inthe nucleic acids, wherein the one or more bridging oligonucleotides arecomplementary to a region of the nucleic acids between the regionscomplementary to the fixed sequence oligonucleotides of each set.

In another general aspect, the assay system employs a method fordetermining the presence or absence of a chromosomal abnormalityassociated with CNV in a genomic region, comprising the steps ofamplifying one or more selected loci from a first chromosome of interestin a mixed sample; amplifying one or more selected loci from a secondchromosome of interest in the mixed sample, determining the relativefrequency of the selected regions from the first and second chromosomesof interest, comparing the relative frequency of the selected regionsfrom the first and second chromosomes of interest, and identifying thepresence or absence of an abnormality based on the compared relativefrequencies of the selected regions. Preferably, two or more nucleicacids regions are selected from each chromosome, and more preferablyfive or more loci are selected from each chromosome.

In yet another general aspect, the assay system employs a method fordetermination of the presence or absence of an aneuploidy in a mixedsample from an individual, comprising the steps of amplifying two ormore selected loci in the cfDNA corresponding to a first chromosome ofinterest in a mixed sample; amplifying two or more selected loci in thecfDNA corresponding to a second chromosome of interest in the mixedsample, determining the relative frequency of the selected regions fromthe first and second chromosomes of interest, comparing the relativefrequency of the selected regions from the first and second chromosomesof interest, and identifying the presence or absence of an aneuploidybased on the compared relative frequencies of the selected regions. In aspecific aspect, the loci of the first and second chromosomes areamplified in a single reaction, and preferably in a single reactioncontained within a single vessel.

Preferably, the assay system detects the presence or absence of loci insamples that can be easily obtained from a subject, such as blood,plasma, serum and the like. In one general aspect, the assay systemutilizes detection of selected regions in cfDNA in a mixed sample. Inone more specific aspect, the assay system utilizes detection ofselected regions in cfDNA of a mixed sample from an individual toidentify the presence or absence of CNVs in a genomic region and thepresence or absence of a polymorphism in one or more loci. Copy numberwithin a genomic region can be determined based on detection ofquantities of selected loci and comparison to the quantities of selectedloci from another genomic region and/or to the quantities of selectedloci from a reference genomic region. In a particular aspect, the ratioof the frequencies of the nucleic acid are compared to a reference meanratio that has been determined for a statistically significantpopulation of genetically “normal” subjects, i.e. subjects that do nothave a CNV associated with the particular loci interrogated in the assaysystem.

In a preferred aspect of the invention, the amplification productscorresponding to the selected nucleic acids are isolated as individualmolecules for analysis of the selected loci. These individualamplification products are isolated from one another, and preferablyphysically isolated (e.g., on a substrate or in individual vessels). Theindividual molecules may be further amplified following isolation tomake multiple, identical copies of the amplification product, a portionthereof, or a nucleic acid complementary to the amplification product ora portion thereof. The detection of the individual molecules or theamplification product may be done through sequencing.

In some aspects, the ligation product is not amplified, but rather isdirectly detected following hybridization, e.g., using single moleculesequencing techniques.

Thus, in a specific aspect, the invention provides an assay system fordetection of the presence or absence of a chromosomal abnormality andpolymorphisms in one or more loci in a mixed sample using a singleassay, the assay comprising the steps of: introducing a first set offixed sequence oligonucleotides to a mixed sample under conditions thatallow the fixed sequence oligonucleotides to specifically hybridize tocomplementary regions in two or more nucleic acids corresponding to aparticular chromosome; introducing a second set of fixed sequenceoligonucleotides to the mixed sample under conditions that allow thefixed sequence oligonucleotides to specifically hybridize tocomplementary regions in nucleic acids with a putative polymorphism;introducing one or more bridging oligonucleotides under conditions thatallow the bridging oligonucleotides to specifically hybridize tocomplementary regions in the nucleic acids, wherein one or more bridgingoligonucleotides are complementary to a region of the nucleic acidsbetween and immediately adjacent to the regions complementary to thefixed sequence oligonucleotides of each set; ligating the hybridizedoligonucleotides to create contiguous ligation products complementary tothe nucleic acids; amplifying the contiguous ligation products to createamplification products having the sequence of the loci; isolatingindividual amplification products; and analyzing the individualamplification products to determine the sequence of all or part of theindividual amplification products. The analysis of the individualamplification products correlates to the presence or absence of achromosomal abnormality and the genetic status of one or more loci inthe mixed sample. For example, the levels of the nucleic acidscorresponding to a particular chromosome may be compared by also usingnucleic acids corresponding to another chromosome, or they may becompared to reference levels for the chromosome being interrogated.

In a preferred aspect, the individual amplification products areanalyzed through sequence determination. In other aspects, theindividual amplification products are analyzed using hybridizationtechniques.

It is a feature of the present invention that copy number of theselected loci can be detected using non-polymorphic detection methods,i.e., detection methods that are not dependent upon the presence orabsence of a particular polymorphism to identify the selected nucleicacid region. In a preferred aspect, the assay detection systems utilizenon-polymorphic detection methods to “count” the relative numbers ofselected loci present in a mixed sample. These numbers can be utilizedto determine if, statistically, a mixed sample is likely to have a CNVin a genomic region in a major and/or minor source within the mixedsample. Similarly, these numbers can be utilized to determine, ifstatistically, nucleic acids from the major source and/or minor sourcehas one or more polymorphisms. Such information can be used to identifya particular pathology or genetic disorder, to confirm a diagnosis orrecurrence of a disease or disorder, to determine the prognosis of adisease or disorder, to assist in determining potential treatmentoptions, etc.

In some aspects, the methods for determination of copy number variationof multiple selected loci from two or more chromosomes in a sample. Thelevels of the different selected loci corresponding to specificchromosomes can be individually quantified and compared to determine thepresence or absence of a chromosomal aneuploidy in one or more cellsource in a mixed sample. The individually quantified regions mayundergo a normalization calculation or the data may be subjected tooutlier exclusion prior to comparison to determine the presence orabsence of an aneuploidy in a mixed sample.

In other aspects, the relative frequencies of the selected loci are usedto determine a chromosome frequency of the first and second chromosomesof interest, and the presence or absence of an aneuploidy is based onthe compared chromosome frequencies of the first and second chromosomesof interest.

In yet other aspects, the relative frequencies of the selected loci areused to determine a chromosome frequency of a chromosome of interest anda reference chromosome, and the presence or absence of an aneuploidy isbased on the compared chromosome frequencies of the chromosome ofinterest and the reference chromosome.

In one particular aspect, the selected loci are isolated prior todetection. The selected loci can be isolated from the mixed sample usingany means that selectively isolate the particular nucleic acids presentin the mixed sample for analysis, e.g., hybridization, amplification orother form of sequence-based isolation of the nucleic acids from themixed sample. Following isolation, the selected nucleic acids areindividually distributed in a suitable detection format, e.g., on amicroarray or in a flow cell, for determination of the sequence and/orrelative quantities of each selected nucleic acid in the mixed sample.The relative quantities of the detected nucleic acids are indicative ofthe number of copies of chromosomes that correspond to the selectednucleic acids present in the mixed sample.

Following isolation and distribution of the selected nucleic acids in asuitable format, the selected sequences are identified, e.g., throughsequence determination of the selected sequence.

In one specific aspect, the invention provides an assay system fordetection of the presence or absence of a fetal aneuploidy, comprisingthe steps of providing a mixed sample comprising maternal and fetalcfDNA, amplifying two or more selected loci from a first and secondchromosome of interest in the mixed sample, amplifying two or moreselected loci from the first and second chromosome of interest in themixed sample, determining the relative frequency of the selected regionsfrom the chromosomes of interest, comparing the relative frequency ofthe selected loci from the first and second chromosomes of interest, andidentifying the presence or absence of a fetal aneuploidy based on thecompared relative frequencies of the selected loci.

In some specific aspects, the relative frequencies of the loci from agenomic region are individually calculated, and the relative frequenciesof the individual loci are compared to determine the presence or absenceof a chromosomal abnormality. In other specific aspects, the relativefrequencies of the selected loci are used to determine a chromosomefrequency of a first and second chromosome of interest and a referencechromosome, and the copy number variation for the chromosome or agenomic region of the chromosome is based on the compared chromosomefrequencies of the first and second chromosomes of interest.

The mixed sample used for analysis can be obtained or derived from anysample which contains the nucleic acid of interest to be analyzed usingthe assay system of the invention. For example, a mixed sample may befrom any maternal fluid which comprises both maternal and fetal cellfree nucleic acids, including but not limited to maternal plasma,maternal serum, or maternal blood. A mixed sample from a transplantpatient would be any fluid or tissue which contains cell free nucleicacids from both the donor cells and the cells of the patient. A mixedsample from a patient with a malignancy would contain cell free nucleicacids from the patient's normal, healthy tissue as well as cell freenucleic acids from the cancerous cells.

Although preferably the assay system is used to detect cfDNA in a mixedsample, in certain aspects the DNA of interest to be analyzed using theassay system of the invention comprises DNA directly from the differentcell types rather than from a mixed sample containing DNA from the majorand minor cell types. Such samples can be obtained from various sourcesdepending upon the target DNA. For example, fetal cells for analysis canbe derived from samples such as amniotic fluid, placenta (e.g., thechorionic villi), and the like. Samples of donor organs can be obtainedin an individual by biopsy. Infectious organisms can be isolateddirectly from an individual and analyzed following isolation. DNA can beextracted from cancerous cells or tissues and used for analysis.

It is a feature of the invention that the nucleic acids analyzed in theassay system do not require polymorphic differences between the cellsources to determine frequency, and thus potential CNVs, in loci from amixed sample. It is another feature of the invention that thesubstantial majority of the nucleic acids isolated from the mixed sampleand detected in the assay system provide information relevant to thepresence, quantity and/or polymorphic nature of a particular locus inthe mixed sample. This ensures that the majority of nucleic acidsanalyzed in the assay system of the invention are informative.

In some aspects, multiple loci are determined for each genomic regionunder interrogation, and the quantity of the selected regions present inthe mixed sample are individually summed to determine the relativefrequency of a locus in a mixed sample. This includes determination ofthe frequency of the locus for the combined maternal and fetal DNApresent in the mixed sample. Preferably, the determination does notrequire a distinction between the DNA from separate sources, although incertain aspects this information may be obtained in addition to theinformation of relative frequencies in the sample as a whole.

In preferred aspects, selected nucleic acids corresponding toinformative loci are detected and summed to determine the relativefrequency of a genomic region in the mixed sample. Frequencies that arehigher than expected for loci from a first genomic region when comparedto the loci from a second locus in a mixed sample is indicative of a CNVof the first genomic region in the mixed sample.

Comparison of genomic regions can be a comparison of part or all of achromosome. For example, the genomic region detected for CNV may be anentire chromosome in the fetus (e.g., chromosomes 18 and 21), where thelikelihood of both being aneuploid is minimal. This can also be acomparison of chromosomes where one is putatively aneuploid (e.g.,chromosome 21) and the other acts as a reference chromosome (e.g. anautosome such as chromosome 2). In yet other aspects, the comparison mayutilize two or more chromosomes that are putatively aneuploid and one ormore reference chromosomes.

In one aspect, the assay system of the invention analyzes multiplenucleic acids representing selected loci on chromosomes of interest, andthe relative frequency of each selected locus from the sample isanalyzed to determine a relative chromosome frequency for eachparticular chromosome of interest in the sample. The chromosomalfrequency of two or more chromosomes or portions thereof is thencompared to statistically determine whether a chromosomal abnormalityexists.

In another aspect, the assay system of the invention analyzes multiplenucleic acids representing selected loci on chromosomes of interest, andthe relative frequency of each selected nucleic acid from the sample isanalyzed and independently quantified to determine a relative amount foreach selected locus in the sample. The sum of the loci in the sample iscompared to statistically determine whether a CNV exists for one or moreloci in a genomic region of one cell source in a mixed sample.

In another aspect, subsets of loci on each chromosome are analyzed todetermine whether a chromosomal abnormality exists. The loci frequencycan be summed for a particular chromosome, and the summations of theloci used to determine aneuploidy. This aspect of the invention sums thefrequencies of the individual loci in each genomic region and thencompares the sum of the loci on a genomic region of one chromosomeagainst a genomic region of another chromosome to determine whether achromosomal abnormality exists. The subsets of loci can be chosenrandomly but with sufficient numbers of loci to yield a statisticallysignificant result in determining whether a chromosomal abnormalityexists. Multiple analyses of different subsets of loci can be performedwithin a mixed sample to yield more statistical power. In anotheraspect, particular loci can be selected that are known to have lessvariation between samples, or by limiting the data used fordetermination of chromosomal frequency, e.g., by ignoring the data fromloci with very high or very low frequency within a sample.

In a particular aspect, the measured quantities of one or moreparticular loci are normalized to account for differences in lociquantity in the sample. This can be done by normalizing for knownvariation from sources such as the assay system (e.g., temperature,reagent lot differences), underlying biology of the sample (e.g.,nucleic acid content), operator differences, or any other variables.

In certain specific aspects, determining the relative percentage ofnucleic acids from the minor source in a mixed sample may be beneficialin performing the assay system, as it will provide important informationon the relative statistical presence of loci that may be indicative ofcopy number variation within the minor source in that sample.Determining the loci contributed to the mixed sample from the minorsource can provide information used to calculate the statisticallysignificant differences in frequencies for genomic regions of interest.Such loci could thus provide two forms of information in theassay—allelic information can be used for determining the percent minorcell contribution in a mixed sample and a summation of the allelicinformation can be used to determine the relative overall frequency ofthat locus in a mixed sample. The allelic information is not needed todetermine the relative overall frequency of that locus.

In another specific aspect, the assay system of the invention can beutilized to determine potential mosaicism in a cell population, andwhether further confirmatory tests should be undertaken to confirm theidentification of mosaicism in the major and/or minor source. In certaininstances, determination of the percent nucleic acids from the minorsource in a mixed sample could assist in quantification of the estimatedlevel of mosaicism. Mosaicism could be subsequently confirmed usingother testing methods that could distinguish mosaic full or partialaneuploidy in specific cells or tissue.

In yet another specific aspect, the assay system of the invention can beutilized to determine contamination in a sample, with the minor speciesrepresenting a contaminant species.

In another aspect, the oligonucleotides for a given selected nucleicacid can be connected at the non-sequence specific ends such that acircular or unimolecular probe may bind thereto. In this aspect, the 3′end and the 5′ end of the circular probe binds to the selected locus andat least one universal amplification region is present in thenon-selected specific sequence of the circular probe.

It is an important feature of the assay that the amplification productsare analyzed directly without the need for enrichment of polymorphicregions from the initial mixed sample. Thus, the current inventionallows detection of both CNV and polymorphisms from a maternal samplewithout an intervening polymorphic enrichment step prior to sequencedetermination of the selected loci.

It is another important feature of the assay that both CNV andpolymorphism detection are determined using a targeted approach ofselected amplification and detection. This allows the majority ofinformation gathered in the assay to be useful for the determination ofthe CNV and/or polymorphism interrogated in the locus of interest, andobviates the need to generate sequence reads that must be aligned with areference sequence.

These and other aspects, features and advantages will be provided inmore detail as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a simplified flow chart of the general steps utilized in theassay systems of the invention.

FIG. 2 illustrates a first general schematic for a ligation-based assaysystem of the invention.

FIG. 3 illustrates a second general schematic for a ligation-based assaysystem of the invention.

FIG. 4 is a third general schematic for a ligation-based assay system ofthe invention.

FIG. 5 illustrates the genotyping performance that was obtained usingone assay system of the invention.

FIG. 6 illustrates the elements used for a detection of aneuploidy andpolymorphism for two cohorts of maternal samples.

FIG. 7 is a summary of patient and sample information and data for asubset of a second cohort of pregnant subjects.

FIG. 8 illustrates the chromosome 21 aneuploidy detection achieved usingone aspect of the invention for a first cohort.

FIG. 9 illustrates the chromosome 18 aneuploidy detection achieved usingone aspect of the invention for a first cohort.

FIG. 10 illustrates the chromosome 21 aneuploidy detection achievedusing one aspect of the invention for a second cohort.

FIG. 11 illustrates the chromosome 18 aneuploidy detection achievedusing one aspect of the invention for a second cohort.

DEFINITIONS

The terms used herein are intended to have the plain and ordinarymeaning as understood by those of ordinary skill in the art. Thefollowing definitions are intended to aid the reader in understandingthe present invention, but are not intended to vary or otherwise limitthe meaning of such terms unless specifically indicated.

The term “amplified nucleic acid” is any nucleic acid molecule whoseamount has been increased at least two fold by any nucleic acidamplification or replication method performed in vitro as compared toits starting amount in a mixed sample.

The term “amplification product” as used herein refers to the productresulting from an amplification reaction using the contiguous ligationproduct as a template, or the product resulting from an amplificationreaction using a molecule complementary to the contiguous ligationproduct as a template.

The term “chromosomal abnormality” refers to any genetic variation thataffects all or part of a chromosome larger than a single locus. Thegenetic variants may include but not be limited to any CNV such asduplications or deletions, translocations, inversions, and mutations.Examples of chromosomal abnormalities include, but are not limited to,Down Syndrome (Trisomy 21), Edwards Syndrome (Trisomy 18), PatauSyndrome (Trisomy 13), Klinefelter's Syndrome (XXY), Triple X syndrome,XYY syndrome, Trisomy 8, Trisomy 16, Turner Syndrome, Robertsoniantranslocation, DiGeorge Syndrome and Wolf-Hirschhorn Syndrome.

The terms “complementary” or “complementarity” are used in reference tonucleic acid molecules (i.e., a sequence of nucleotides) that arerelated by base-pairing rules. Complementary nucleotides are, generally,A and T (or A and U), or C and G. Two single stranded RNA or DNAmolecules are said to be substantially complementary when thenucleotides of one strand, optimally aligned and with appropriatenucleotide insertions or deletions, pair with at least about 90% toabout 95% complementarity, and more preferably from about 98% to about100% complementarity, and even more preferably with 100%complementarity. Alternatively, substantial complementarity exists whenan RNA or DNA strand will hybridize under selective hybridizationconditions to its complement. Selective hybridization conditionsinclude, but are not limited to, stringent hybridization conditions.Stringent hybridization conditions will typically include saltconcentrations of less than about 1 M, more usually less than about 500mM and preferably less than about 200 mM. Hybridization temperatures aregenerally at least about 2° C. to about 6° C. lower than meltingtemperatures (T_(m)).

The term “copy number variation” or “CNV” as used interchangeably hereinare alterations of the DNA of a genome that results in a cell having anabnormal number of copies of one or more loci in the DNA. CNVs that areclinically relevant can be limited to a single gene or include acontiguous set of genes. A CNV can also correspond to relatively largeregions of the genome that have been deleted, inverted or duplicated oncertain chromosomes, up to an including one or more additional copies ofa complete chromosome. The term CNV as used herein does not refer to anysequence-related information, but rather to quantity or “counts” ofgenetic regions present in a sample.

The term “correction index” refers to an index that may containadditional nucleotides that allow for identification and correction ofamplification, sequencing or other experimental errors including thedetection of deletion, substitution, or insertion of one or more basesduring sequencing as well as nucleotide changes that may occur outsideof sequencing such as oligo synthesis, amplification, and any otheraspect of the assay. These correction indices may be stand-alone indicesthat are separate sequences, or they may be embedded within otherregions to assist in confirming accuracy of the experimental techniquesused, e.g., a correction index may be a subset of sequences used foruniversal amplification or a subset of nucleotides of a sample locus.

The term “diagnostic tool” as used herein refers to any composition orassay of the invention used in combination as, for example, in a systemin order to carry out a diagnostic test or assay on a patient sample.

The term “disease trait” refers to a monogenic or polygenic traitassociated with a pathological condition, e.g., a disease, disorder,syndrome or predisposition.

The term “hybridization” generally means the reaction by which thepairing of complementary strands of nucleic acid occurs. DNA is usuallydouble-stranded, and when the strands are separated they willre-hybridize under the appropriate conditions. Hybrids can form betweenDNA-DNA, DNA-RNA or RNA-RNA. They can form between a short strand and along strand containing a region complementary to the short one.Imperfect hybrids can also form, but the more imperfect they are, theless stable they will be (and the less likely to form).

The term “informative locus” as used herein refers to a locus that ishomozygous for one cell source and heterozygous for a second cell sourceon a particular chromosome or portion of a chromosome interrogated forpurposes of determining a CNV of all or part of that chromosome.Informative loci for use in the assay system of the invention includeloci used for interrogation of a reference chromosome as well as lociused for interrogation of a chromosome that is putatively aneuploid in acell source. Informative loci can also distinguish copy number of lociin cell sources from different individuals within a single individual(e.g., detection of transplant donor cells in a transplant recipient ordetection of a fetal DNA within a maternal mixed sample).

The terms “locus” and “loci” as used herein refer to a locus of knownlocation in a genome.

The term “major source” refers to a source of nucleic acids in a samplefrom an individual that is representative of the predominant genomicmaterial in that individual.

The term “maternal sample” as used herein refers to any sample takenfrom a pregnant mammal which comprises both fetal and maternal cell freegenomic material (e.g., DNA). Preferably, maternal samples for use inthe invention are obtained through relatively non-invasive means, e.g.,phlebotomy or other standard techniques for extracting peripheralsamples from a subject.

The term “melting temperature” or T_(m) is commonly defined as thetemperature at which a population of double-stranded nucleic acidmolecules becomes half dissociated into single strands. The equation forcalculating the T_(m) of nucleic acids is well known in the art. Asindicated by standard references, a simple estimate of the T_(m) valuemay be calculated by the equation: T_(m)=81.5+16.6(log10[Na+])0.41(%[G+C])−675/n−1.0 m, when a nucleic acid is in aqueoussolution having cation concentrations of 0.5 M or less, the (G+C)content is between 30% and 70%, n is the number of bases, and m is thepercentage of base pair mismatches (see, e.g., Sambrook J et al.,Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press (2001)). Other references include more sophisticatedcomputations, which take structural as well as sequence characteristicsinto account for the calculation of T_(m).

“Microarray” or “array” refers to a solid phase support having asurface, preferably but not exclusively a planar or substantially planarsurface, which carries an array of sites containing nucleic acids suchthat each site of the array comprises substantially identical oridentical copies of oligonucleotides or polynucleotides and is spatiallydefined and not overlapping with other member sites of the array; thatis, the sites are spatially discrete. The array or microarray can alsocomprise a non-planar interrogatable structure with a surface such as abead or a well. The oligonucleotides or polynucleotides of the array maybe covalently bound to the solid support, or may be non-covalentlybound. Conventional microarray technology is reviewed in, e.g., Schena,Ed., Microarrays: A Practical Approach, IRL Press, Oxford (2000). “Arrayanalysis”, “analysis by array” or “analysis by microarray” refers toanalysis, such as, e.g., sequence analysis, of one or more biologicalmolecules using a microarray.

The term “minor source” refers to a source of nucleic acids within anindividual that is present in limited amounts and which isdistinguishable from the major source due to differences in its genomicmakeup and/or expression. Examples of minor sources include, but are notlimited to, fetal cells in a pregnant female, cancerous cells in apatient with a malignancy, cells from a donor organ in a transplantpatient, nucleic acids from an infectious organism in an infected host,and the like.

The term “mixed sample” as used herein refers to any sample comprisingcell free genomic material (e.g., DNA) from two or more cell types ofinterest, one being a major source and the other being a minor sourcewithin a single individual. Mixed samples include samples with genomicmaterial from both a major and a minor source in an individual, whichmay be e.g., normal and atypical somatic cells, or cells that comprisegenomes from two different individuals, e.g., a sample with bothmaternal and fetal genomic material or a sample from a transplantpatient that comprises cells from both the donor and recipient. Mixedsamples are preferably peripherally derived, e.g., from blood, plasma,serum, etc.

The term “monogenic trait” as used herein refers to any trait, normal orpathological, that is associated with a mutation or polymorphism in asingle gene. Such traits include traits associated with a disease,disorder, or predisposition caused by a dysfunction in a single gene.Traits also include non-pathological characteristics (e.g., presence orabsence of cell surface molecules on a specific cell type).

The term “non-maternal” allele means an allele with a polymorphismand/or mutation that is found in a fetal allele (e.g., an allele with ade novo SNP or mutation) and/or a paternal allele, but which is notfound in the maternal allele.

By “non-polymorphic”, when used with respect to detection of selectedloci, is meant a detection of such locus, which may contain one or morepolymorphisms, but in which the detection is not reliant on detection ofthe specific polymorphism within the region. Thus a selected locus maycontain a polymorphism, but detection of the region using the assaysystem of the invention is based on occurrence of the region rather thanthe presence or absence of a particular polymorphism in that region.

As used herein “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes ribonucleoside triphosphatesATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP,dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivativesinclude, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, andnucleotide derivatives that confer nuclease resistance on the nucleicacid molecule containing them. The term nucleotide as used herein alsorefers to dideoxyribonucleoside triphosphates (ddNTPs) and theirderivatives. Illustrated examples of dideoxyribonucleoside triphosphatesinclude, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.

According to the present invention, a “nucleotide” may be unlabeled ordetectably labeled by well known techniques. Fluorescent labels andtheir attachment to oligonucleotides are described in many reviews,including Haugland, Handbook of Fluorescent Probes and ResearchChemicals, 9th Ed., Molecular Probes, Inc., Eugene Oreg. (2002); Kellerand Manak, DNA Probes, 2nd Ed., Stockton Press, New York (1993);Eckstein, Ed., Oligonucleotides and Analogues: A Practical Approach, IRLPress, Oxford (1991); Wetmur, Critical Reviews in Biochemistry andMolecular Biology, 26:227-259 (1991); and the like. Other methodologiesapplicable to the invention are disclosed in the following sample ofreferences: Fung et al., U.S. Pat. No. 4,757,141; Hobbs, Jr., et al.,U.S. Pat. No. 5,151,507; Cruickshank, U.S. Pat. No. 5,091,519; Menchenet al., U.S. Pat. No. 5,188,934; Begot et al., U.S. Pat. No. 5,366,860;Lee et al., U.S. Pat. No. 5,847,162; Khanna et al., U.S. Pat. No.4,318,846; Lee et al., U.S. Pat. No. 5,800,996; Lee et al., U.S. Pat.No. 5,066,580: Mathies et al., U.S. Pat. No. 5,688,648; and the like.Labeling can also be carried out with quantum dots, as disclosed in thefollowing patents and patent publications: U.S. Pat. Nos. 6,322,901;6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143;5,990,479; 6,207,392; 2002/0045045; and 2003/0017264. Detectable labelsinclude, for example, radioactive isotopes, fluorescent labels,chemiluminescent labels, bioluminescent labels and enzyme labels.Fluorescent labels of nucleotides may include but are not limitedfluorescein, 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanineand 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specificexamples of fluorescently labeled nucleotides include [R6G]dUTP,[TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP,[FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP,[dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from PerkinElmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLinkCy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLinkCy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, ArlingtonHeights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP,Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP,Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from BoehringerMannheim, Indianapolis, Ind.; and Chromosomee Labeled Nucleotides,BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP,BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, CascadeBlue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP,fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP,Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP,tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, andTexas Red-12-dUTP available from Molecular Probes, Eugene, Oreg.

The terms “oligonucleotides” or “oligos” as used herein refer to linearoligomers of natural or modified nucleic acid monomers, includingdeoxyribonucleotides, ribonucleotides, anomeric forms thereof, peptidenucleic acid monomers (PNAs), locked nucleotide acid monomers (LNA), andthe like, or a combination thereof, capable of specifically binding to asingle-stranded polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Usually monomers are linked by phosphodiesterbonds or analogs thereof to form oligonucleotides ranging in size from afew monomeric units, e.g., 8-12, to several tens of monomeric units,e.g., 100-200 or more. Suitable nucleic acid molecules may be preparedby the phosphoramidite method described by Beaucage and Carruthers(Tetrahedron Lett., 22:1859-1862 (1981)), or by the triester methodaccording to Matteucci, et al. (J. Am. Chem. Soc., 103:3185 (1981)),both incorporated herein by reference, or by other chemical methods suchas using a commercial automated oligonucleotide synthesizer.

The term “polygenic trait” as used herein refers to any trait, normal orpathological, that is associated with a mutation or polymorphism in morethan a single gene. Such traits include traits associated with adisease, disorder, syndrome or predisposition caused by a dysfunction intwo or more genes. Traits also include non-pathological characteristicsassociated with the interaction of two or more genes.

As used herein the term “polymerase” refers to an enzyme that linksindividual nucleotides together into a long strand, using another strandas a template. There are two general types of polymerase—DNApolymerases, which synthesize DNA, and RNA polymerases, which synthesizeRNA. Within these two classes, there are numerous sub-types ofpolymerases, depending on what type of nucleic acid can function astemplate and what type of nucleic acid is formed.

As used herein “polymerase chain reaction” or “PCR” refers to atechnique for replicating a specific piece of selected DNA in vitro,even in the presence of excess non-specific DNA. Primers are added tothe selected DNA, where the primers initiate the copying of the selectedDNA using nucleotides and, typically, Taq polymerase or the like. Bycycling the temperature, the selected DNA is repetitively denatured andcopied. A single copy of the selected DNA, even if mixed in with other,random DNA, can be amplified to obtain billions of replicates. Thepolymerase chain reaction can be used to detect and measure very smallamounts of DNA and to create customized pieces of DNA. In someinstances, linear amplification methods may be used as an alternative toPCR.

The term “polymorphism” as used herein refers to any genetic changes orsequence variants in a locus, including but not limited to singlenucleotide polymorphisms (SNPs), methylation differences, short tandemrepeats (STRs), single gene polymorphisms, point mutations,trinucleotide repeats, indels and the like.

Generally, a “primer” is an oligonucleotide used to, e.g., prime DNAextension, ligation and/or synthesis, such as in the synthesis step ofthe polymerase chain reaction or in the primer extension techniques usedin certain sequencing reactions. A primer may also be used inhybridization techniques as a means to provide complementarity of alocus to a capture oligonucleotide for detection of a specific locus.

The term “research tool” as used herein refers to any composition orassay of the invention used for scientific enquiry, academic orcommercial in nature, including the development of pharmaceutical and/orbiological therapeutics. The research tools of the invention are notintended to be therapeutic or to be subject to regulatory approval;rather, the research tools of the invention are intended to facilitateresearch and aid in such development activities, including anyactivities performed with the intention to produce information tosupport a regulatory submission.

The term “sample index” refers generally to a series of uniquenucleotides (i.e., each sample index is unique to a sample in amultiplexed assay system for analysis of multiple samples). The sampleindex can thus be used to assist in locus identification formultiplexing of different samples in a single reaction vessel, such thateach sample can be identified based on its sample index. In a preferredaspect, there is a unique sample index for each sample in a set ofsamples, and the samples are pooled during sequencing. For example, iftwelve samples are pooled into a single sequencing reaction, there areat least twelve unique sample indexes such that each sample is labeleduniquely.

The term “selected locus” as used herein refers to a locus correspondingto a loci interrogated, e.g., for copy number, the presence or absenceof one or more polymorphism, presence or absence of an infectiousorganism, etc. Such selected loci may be directly isolated and amplifiedfrom the sample for detection, e.g., based on hybridization and/or othersequence-based techniques, or they may be amplified using the sample asa template prior to detection of the sequence. Nucleic acids regions foruse in the assay systems of the present invention may be selected on thebasis of DNA level variation between individuals, based upon specificityfor a particular chromosome, based on CG content and/or requiredamplification conditions of the selected loci, or other characteristicsthat will be apparent to one skilled in the art upon reading the presentdisclosure.

The terms “sequencing”, “sequence determination” and the like as usedherein refers generally to any and all biochemical methods that may beused to determine the order of nucleotide bases in a nucleic acid.

The term “specifically binds”, “specific binding” and the like as usedherein, when referring to a binding partner (e.g., a nucleic acid probeor primer, antibody, etc.) that results in the generation of astatistically significant positive signal under the designated assayconditions. Typically the interaction will subsequently result in adetectable signal that is at least twice the standard deviation of anysignal generated as a result of undesired interactions (background).

The term “status” as used herein in relationship to a gene refers to thesequence status of the alleles of a particular gene, including thecoding regions and the non-coding regions that affect the translationand/or protein expression from that gene. The status of a geneassociated with an autosomal dominant disease such as achondroplasia(e.g., the gene encoding the fibroblast growth factor receptor) orHuntington's disease (e.g., the Huntingtin gene), or for an X-linkeddisease in the case of a male fetus, can be classified as affected i.e.,one allele possesses mutation(s) that is causative of the diseases ordisorder, or non-affected, i.e. both alleles lack such mutations(s). Thestatus of a gene associated with an autosomal recessive disease or amaternal gene associated with an X-linked recessive disorder, may beclassified as affected, i.e., both alleles possess mutation(s) causativeof the diseases or disorder; carrier, i.e. one allele possessesmutation(s) causative of the diseases or disorder; or non-affected, i.e.both alleles lack such mutations(s). The status of a gene may alsoindicate the presence or absence of a particular allele associated witha risk of developing a polygenic disease, e.g., a polymorphism that isprotective against a particular disease or disorder or a polymorphismassociated with an enhanced risk for a particular disease or disorder.

DETAILED DESCRIPTION OF THE INVENTION

The assay systems and methods described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, microarray and sequencing technology, which are within theskill of those who practice in the art. Such conventional techniquesinclude polymer array synthesis, hybridization and ligation ofoligonucleotides, sequencing of oligonucleotides, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,equivalent conventional procedures can, of course, also be used. Suchconventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds.,Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler,Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNAMicroarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics:Sequence and Genome Analysis (2004); Sambrook and Russell, CondensedProtocols from Molecular Cloning: A Laboratory Manual (2006); andSambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (allfrom Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4thEd.) W.H. Freeman, New York (1995); Gait, “Oligonucleotide Synthesis: APractical Approach” IRL Press, London (1984); Nelson and Cox, Lehninger,Principles of Biochemistry, 3^(rd) Ed., W. H. Freeman Pub., New York(2000); and Berg et al., Biochemistry, 5^(th)Ed., W.H. Freeman Pub., NewYork (2002), all of which are herein incorporated by reference in theirentirety for all purposes. Before the present compositions, researchtools and methods are described, it is to be understood that thisinvention is not limited to the specific methods, compositions, targetsand uses described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to limit thescope of the present invention, which will be limited only by appendedclaims.

It should be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “alocus” refers to one, more than one, or mixtures of such regions, andreference to “an assay” includes reference to equivalent steps andmethods known to those skilled in the art, and so forth.

Where a range of values is provided, it is to be understood that eachintervening value between the upper and lower limit of that range—andany other stated or intervening value in that stated range—isencompassed within the invention. Where the stated range includes upperand lower limits, ranges excluding either of those included limits arealso included in the invention.

Unless expressly stated, the terms used herein are intended to have theplain and ordinary meaning as understood by those of ordinary skill inthe art. The following definitions are intended to aid the reader inunderstanding the present invention, but are not intended to vary orotherwise limit the meaning of such terms unless specifically indicated.All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing the formulations and methodologiesthat are described in the publication and which might be used inconnection with the presently described invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

The Invention in General

The present invention provides single assay systems with the ability todetect copy number variations, polymorphisms and infectious disease in amixed sample from a single individual. The assay methods compriseidentifying the copy number of selected loci (including multiple locithat are associated with chromosomal abnormalities) and detection ofpolymorphisms that are found in a major source and/or a minor source.These methods are useful for any mixed sample containing genomicmaterial (e.g., DNA) from a major and a minor source that are present ina single individual.

The use of selected loci in the assay methods of the invention providesamplification of loci for detection of copy number variation. A distinctadvantage of the invention is that the selected loci corresponding tocopy number variation and/or polymorphisms can be further analyzed usinga variety of detection and quantification techniques, including but notlimited to hybridization techniques, digital PCR and high throughputsequencing determination techniques. Selection probes can be designedagainst any number of loci for any chromosome. Although amplification ofthe mixed sample prior to the identification and quantification of theselection nucleic acids regions is not mandatory, limited amplificationprior to detection can be performed, in particular if the initialamounts of nucleic acid are limited.

FIG. 1 is a simplified flow chart of the general steps utilized in theassay systems of the invention. FIG. 1 shows method 100, where in afirst step 110, a mixed nucleic acid sample is provided for analysis.The mixed sample can be prepared from virtually any sample as suchtechniques are known to those of skill in the art (see, e.g., TietzTextbook of Clinical Chemistry and Molecular Diagnostics, 4th Ed.,Chapter 2, Burtis, C. Ashwood E. and Bruns, D, eds. (2006); ChemicalWeapons Convention Chemicals Analysis: Sample Collection, Preparationand Analytical Methods, Mesilaakso, M., ed., (2005); Pawliszyn, J.,Sampling and Sample Preparation for Field and Laboratory, (2002);Venkatesh Iyengar, G., et al., Element Analysis of Biological Samples:Principles and Practices (1998); Drielak, S., Hot Zone Forensics:Chemical, Biological, and Radiological Evidence Collection (2004);Wells, D., High Throughput Bioanalytical Sample Preparation (Progress inPharmaceutical and Biomedical Analysis) (2002)), each of which isincorporated by reference). Depending on the type of mixed samplechosen, additional processing and/or purification steps may be performedto obtain nucleic acid fragments of a desired purity or size, usingprocessing methods including but not limited to sonication,nebulization, gel purification, PCR purification systems, nucleasecleavage, or a combination of these methods. In a preferred aspect,samples comprising cell-free DNA (cfDNA) are used.

At step 120, a first set of fixed sequence oligonucleotides areintroduced to the mixed nucleic acid sample, under conditions that allowthe first set of fixed sequence oligonucleotides to hybridize to themixed nucleic acid sample. The first set of fixed sequenceoligonucleotides comprise nucleic acid sequences that are complementaryto a selected locus or loci in the mixed sample, which as will bedescribed in detail herein are useful in determining copy numbervariations and/or chromosomal abnormalities. The nucleic acid sequencescapable of determining copy number variations and/or chromosomalabnormalities include sequences that allow for identification ofchromosomal abnormalities such as duplications or deletions,aneuploidies, translocations, or inversions.

At step 130, a second set of fixed sequence oligonucleotides areintroduced to the mixed nucleic acid sample and first set of fixedsequence oligonucleotides under conditions that allow the second set offixed sequence oligonucleotides to hybridize to the mixed nucleic acidsample. The second set of fixed sequence oligonucleotides comprisenucleic acid sequences that are complementary to a selected locus orloci in the mixed sample, able to detect polymorphisms. Washing stepsoptionally may be included between steps 120 and 130, and 130 and 140.

Although the invention is described as the two sets of oligos introducedto the mixed sample sequentially, the order of the sets may be reversedfrom that described in the figures or, in preferred aspects, they can beintroduced simultaneously.

At step 140, first and second sets of fixed sequence oligonucleotidesthat have hybridized to adjacent regions of the selected loci in themixed sample are ligated, and at step 150, the ligated oligonucleotidesare amplified. The ligated and amplified oligonucleotides are thendetected and analyzed, which allows for determination of copy numbervariations or chromosomal abnormalities and identification ofpolymorphisms at step 160.

The sets of fixed sequence nucleic acids are designed to hybridize to atleast two separate regions in a selected locus. In preferred aspects,two or more separate oligos are used to hybridize to these regions toprovide adjacent nucleic acids complementary to the selected locus. Insome aspects, however, a single probe can be used which comprises two ormore distinct non-adjacent regions that are complementary to theselected loci including precircular probes such as so-called “padlockprobes” or “molecular inversion probes (MIPs)”.

The present invention provides an improved system over more randomtechniques such as massively parallel sequencing, shotgun sequencing,and the use of random digital PCR which have been used by others todetect CNVs. These aforementioned approaches rely upon sequencing of allor a statistically significant population of DNA fragments in a sample,followed by mapping of these fragments or otherwise associating thefragments to their appropriate chromosomes. The identified fragments arethen compared against each other or against some other reference (e.g.,normal chromosomal makeup) to determine CNVs on particular chromosomes.These methods are inherently inefficient as compared to the presentinvention, as the primary chromosomes of interest only constitute aminority of data that is generated from the detection of such DNAfragments in the mixed samples.

The assays of the present invention provide targeted detection ofselected loci, which provides information on both the content of theselected region (i.e., presence of a polymorphic region) and informationon the frequency of the detected region in a sample (with or withoutdetecting any putative polymorphisms in that region). This key featureprovides the ability to detect both copy number of selected regions andthe presence or absence of polymorphisms in a selected region as asingle data set from performance of a multiplexed assay of theinvention.

Techniques that are dependent upon a very broad sampling of DNA in asample provide a very broad coverage of the DNA analyzed, but in factare sampling the DNA contained within a sample on a 1× or less basis(i.e., subsampling). In contrast, the selective amplification used inthe present assays are specifically designed to provide depth ofcoverage of particular loci of interest, and provide a “super-sampling”of such selected loci with an average sequence coverage of preferably 2×or more, more preferably sequence coverage of 100× of more, even morepreferably sequence coverage of 1000× or more of the selected loci(including from the one or more minor sources) present in the initialmixed sample.

A distinct advantage of the invention is that the ligation productsresulting from the assays corresponding to chromosomal abnormalitiesand/or chromosomal abnormalities and polymorphisms can be analyzed usinga variety of detection and quantification techniques, including but notlimited to hybridization techniques, digital PCR and high throughputsequencing determination techniques.

The assay systems of the invention provide a more efficient andeconomical use of data, and the substantial majority of sequencesanalyzed following sample amplification result in affirmativeinformation about the presence of a particular CNV in the mixed sample.Thus, unlike techniques relying on massively parallel sequencing orrandom digital “counting” of chromosome regions and subsequentidentification of relevant data from such counts, the assay system ofthe invention provides a much more efficient use of data collection thanthe random approaches taught by others in the art.

Assay Methods

The assay systems of the invention utilize a general scheme as describedabove, though many different configurations and variations can beemployed, a few of which are described below and more of which areexemplified in U.S. Ser. No. 61/371,605 filed Aug. 6, 2010, and U.S.Ser. No. 13/013,732, both of which are incorporated by reference hereinin their entirety.

FIG. 2 illustrates a first general schematic for a ligation-based assaysystem of the invention. The fixed sequence oligonucleotides 201, 203comprise universal primer regions 209 and 211, respectively, and regionscomplementary to the selected locus 205 and 207, respectively. However,in addition, the assay system in FIG. 2 employs a sample index region221 on the first fixed sequence oligonucleotide 201. In certain aspects,all or a portion of the sequences of the selected loci are directlydetected using the described techniques, e.g., by sequence determinationor hybridization techniques. In the example of FIG. 2, a sample index isassociated with the first fixed sequence oligonucleotide 201. Thedetection of the indices can identify a sequence from a specific samplein a highly multiplexed assay system.

At step 202, the fixed sequence oligonucleotides 201, 203 are introducedin step 202 to the mixed sample 200 and allowed to specifically bind tothe selected locus 215. Following hybridization, the unhybridized fixedsequence oligonucleotides are preferably separated from the remainder ofthe genetic sample (by, e.g., washing—not shown). A bridging oligo isthen introduced and allowed to hybridize in step 204 to the region ofthe locus 215 between the first 201 and second 203 fixed sequenceoligonucleotides. The bound oligonucleotides are ligated at step 206 tocreate a contiguous nucleic acid spanning and complementary to the locusof interest. In certain aspects of the invention, the bridgingoligonucleotides of are between 2-45 nucleotides in length. In aspecific aspect, the bridging oligonucleotides are between 3-9nucleotides in length. In yet another specific aspect, the bridgingoligonucleotides are between 10-30 nucleotides in length.

Following ligation, the ligation product is eluted from the gDNAtemplate. Universal primers 217, 219 are introduced in step 208 toamplify the ligated first and second fixed sequence oligonucleotides tocreate 210 amplification products 223 that comprise the sequence of thelocus of interest. These products 223 are isolated, detected, identifiedand quantified to provide information regarding the presence and amountof the selected loci in the mixed sample. Preferably, the amplificationproducts are detected and quantified through sequence determination. Inspecific aspects, it is desirable to determine the sequences of both theindex and the amplification products, for example, to provideidentification of the sample as well as the locus. The indicesenvisioned in the invention may be associated with the first fixedsequence oligonucleotides, the second fixed sequence oligonucleotides orboth. Alternatively or in addition, indices may be associated withprimers that are used to amplify the ligated first and second fixedsequence oligonucleotides, which also serves to incorporate indices intothe amplification products.

In preferred aspects, indices representative of the mixed sample fromwhich a nucleic acid may be isolated are used to identify the source ofthe selected loci in a multiplexed assay system. In such aspects, thenucleic acids are uniquely identified with the sample index. Uniquelyidentified oligonucleotides may then be combined into a single reactionvessel with nucleic acids from other mixed samples prior to sequencing.In such a case, the sequencing data is segregated by the unique sampleindex to determine the frequency of each target locus for each mixedsample and to determine whether there is a chromosomal abnormality in anindividual sample.

In aspects of the invention using sample indices, the fixed sequenceoligonucleotides preferably are designed so that sample indicescomprising identifying information are located between the universalprimer regions 209 and 211 and the regions complementary to the selectedloci in the sample 205 and 207. Alternatively, the indices and universalamplification sequences can be added to the ligated first and secondfixed sequence oligos (and the bridging oligo, if present) by includingthese indices in the primers used to amplify the ligation products forseparate samples. In either case, preferably the indices are encodedupstream of the locus-specific sequences but downstream of the universalprimers so that they are preserved upon amplification.

FIG. 3 exemplifies methods of the assay system in which one or morebridging oligonucleotides are employed and exemplifies how polymorphismsmay be detected and identified. In FIG. 3, two fixed sets of sequenceoligonucleotides are used which comprise substantially the sameuniversal primers 309, 311 and sequence-specific regions 305, 307, butcomprise different sample indices, 321, 323 on the fixed sequenceoligonucleotides of the set where the different indices correspond todifferent base sequences for the single nucleotide polymorphism presentin a particular sample. The ligation reactions are carried out withmaterial from the same mixed sample 300, but in separate tubes with thedifferent allele-specific oligo sets. Bridging oligonucleotidescorresponding to two possible nucleotides for this SNP in the selectedloci 313, 333 are used to detect of the selected locus in each ligationreaction. Two allele indices 321, 323 that are indicative of theparticular polymorphic alleles are incorporated into the amplificationproducts so that sequence determination of the actual sequence of theligated first, second and bridging oligonucleotides are not necessarilyneeded, although the sequences of the entire ligation products may stillbe determined to identify and/or provide confirmation.

Each of the fixed sequence oligonucleotides comprises a regioncomplementary to the selected locus 305, 307, and universal primerregions 309, 311 used to amplify the different selected loci followinginitial selection and/or isolation of the selected loci from the mixedsample. The universal primer regions are located at the ends of thefixed sequence oligonucleotides 301, 303, and 323 flanking the indicesand the regions complementary to the nucleic acid of interest, thuspreserving the nucleic acid-specific sequences and the sample indices inthe products of any universal amplification methods. The fixed sequenceoligonucleotides 301, 303, 323 are introduced at step 302 to an aliquotof the genetic sample 300 and allowed to specifically bind to theselected loci 315 or 325. Following hybridization, the unhybridizedfixed sequence oligonucleotides are preferably separated from theremainder of the genetic sample by, e.g., washing (not shown).

The bridging oligos corresponding to an A/T SNP 313 or a G/C SNP 333 areintroduced and allowed to bind in step 304 to the region of the selectedlocus 315 or 325 between the first 305 and second 307 nucleicacid-complementary regions of the fixed sequence oligonucleotides.Alternatively, the bridging oligos 313, 333 can be introduced to thesample simultaneously with the fixed sequence oligonucleotides. Thebound oligonucleotides are ligated in step 306 in the single reactionmixture to create a contiguous nucleic acid spanning and complementaryto the selected locus.

Following ligation, the separate reactions may preferably be combinedfor the universal amplification and detection steps. Universal primers317, 319 are introduced to the combined reactions at step 308 to amplifythe ligated template regions and create at step 310 ligated first andsecond fixed sequence oligos and bridging oligo products 327, 329 thatcomprise the sequence of the selected locus representing both SNPs inthe selected locus. These ligation products 327, 329 are detected andquantified through sequence determination of the ligation product,through the sample index and/or the region of the product containing theSNP in the selected locus.

In an alternative configuration of the methods of the assay systems ofthe invention, the bridging oligo may hybridize to a region that is notdirectly adjacent to the region complementary to one or both of thefixed sequence oligos, and an intermediate step requiring extension ofone or more of the oligos is necessary prior to ligation. For example,as illustrated in FIG. 4, each set of oligonucleotides preferablycontains two oligonucleotides 401, 403 of fixed sequence and one or morebridging oligonucleotides 413. Each of the fixed sequenceoligonucleotides comprises a region complementary to the selected locus405, 407, and primer sequences, preferably universal primer sequences,409, 411, i.e., oligo regions complementary to universal primers. Theprimer sequences 409, 411 are located at or near the ends of the fixedsequence oligonucleotides 401, 403, and thus preserve the nucleicacid-specific sequences in the products of any universal amplificationmethods. The fixed sequence oligonucleotides 401, 403 are introduced atstep 402 to the mixed sample 400 and allowed to specifically bind to thecomplementary portions of the locus of interest 415. Followinghybridization, the unhybridized fixed sequence oligonucleotides arepreferably separated from the remainder of the genetic sample (notshown).

The bridging oligonucleotide is then introduced at step 404 and allowedto bind to the region of the selected locus 415 between the first 401and second 403 fixed sequence oligonucleotides. Alternatively, thebridging oligo can be introduced simultaneously with the fixed sequenceoligonucleotides. In this exemplary aspect, the bridging oligohybridizes to a region directly adjacent to the first fixed sequenceoligo region 405, but is separated by one or more nucleotides from thecomplementary region of the second fixed sequence oligonucleotide 407.Following hybridization of the fixed sequence and bridging oligos, thebridging oligo 413 is extended at step 406, e.g., using a polymerase anddNTPs, to fill the gap between the bridging oligo 413 and the secondfixed sequence oligo 403. Following extension, the boundoligonucleotides are ligated at step 408 to create a contiguous nucleicacid spanning and complementary to the locus of interest 415. Afterligation, universal primers 417, 419 are introduced at step 410 toamplify the ligated first, second and bridging oligos to create at step412 amplification products 423 that comprise the sequence of theselected locus of interest. Amplification products 423 are optionallyisolated, detected, and quantified to provide information on thepresence and amount of the selected locus(s) in the mixed sample.

Detecting Copy Number Variations

The assay systems utilize nucleic acid probes designed to identify, andpreferably to isolate, selected nucleic acids regions in a mixed sample.Certain of the probes identify sequences of interest in selected lociinterrogated for copy number (i.e. loci frequency), and other probesidentify sequences that correspond to polymorphisms of interest (i.e.loci content) in nucleic acids corresponding to a major source or minorsource in a mixed sample.

In specific aspects, the assay systems of the invention employ one ormore selective amplification steps (e.g., using one or more primers thatspecifically hybridize to a selected locus) for isolating, amplifying oranalyzing substantially all of the selected loci analyzed. This is indirect contrast to the random amplification approach used by othersemploying, e.g., massively parallel sequencing, as such amplificationtechniques generally involve random amplification of all or asubstantial portion of the genome. In addition, although the initialsample can be enriched using methods such as general amplification toincrease the copy number of nucleic acids in the mixed sample,preferably no enrichment steps are used prior to the hybridization,ligation, and amplification steps used to identify the loci of interest.

In a general aspect, the user of the invention analyzes multipleselected loci on different chromosomes. When multiple loci are analyzedfor a sample, a preferred embodiment is to amplify all of the selectedloci for each sample in one reaction vessel. The frequency or amount ofthe multiple selected loci are analyzed to determine whether achromosomal abnormality exists, and the presence or absence of apolymorphism is analyzed to determine the presence or absence orlikelihood calculation of a chromosomal abnormality in a source in themixed sample.

In preferred aspects, multiple selected loci from two or more samplesmay be amplified in a single reaction vessel, and the informationsimultaneously analyzed in a single data set, e.g., through sequencedetermination. The resulting data is then analyzed to separate theresults for the different sample and used to determine the presence ofabsence of CNV and/or the presence of absence of polymorphisms forindividual samples.

In one aspect, chromosomal abnormalities are identified in the assaysystem of the invention using multiple selected loci on multiplechromosomes, and the frequency of the selected loci on the multiplechromosomes compared to identify an increase likelihood of aneuploidybased on the ratios of the chromosomes. Normalization or standardizationof the frequencies can be performed for one or more selected loci.

In another aspect, the assay system sums the frequencies of the selectedloci on two or more chromosomes and then compares the sum of theselected loci on one chromosome against another chromosome to determinewhether a chromosomal aneuploidy exists. In another aspect, the assaysystem analyzes subsets of selected loci on two or more chromosomes todetermine whether a chromosomal aneuploidy exists for one of the twochromosomes. The comparison can be made either within the same ordifferent chromosomes.

In certain aspects, the data used to determine the frequency of theselected loci may exclude outlier data that appear to be due toexperimental error, or that have elevated or depressed levels based onan idiopathic genetic bias within a particular sample. In one example,the data used for summation may exclude DNA regions with a particularlyelevated frequency in one or more samples. In another example, the dataused for summation may exclude selected loci that are found in aparticularly low abundance in one or more samples.

In another aspect subsets of loci can be chosen randomly but withsufficient numbers of loci to yield a statistically significant resultin determining whether a chromosomal abnormality exists. Multipleanalyses of different subsets of loci can be performed within a mixedsample to yield more statistical power. For example, if there are 100selected regions for chromosome 21 and 100 selected regions forchromosome 18, a series of analyses could be performed that evaluatefewer than 100 regions for each of the chromosomes. In this example,selected loci are not being selectively excluded.

The quantity of different nucleic acids detectable on certainchromosomes may vary depending upon a number of factors, includinggeneral representation of loci in different cell sources in maternalsamples, degradation rates of the different nucleic acids representingdifferent loci in mixed samples, sample preparation methods, and thelike. Thus, in another aspect, the quantity of particular loci on achromosome is summed to determine the loci quantity for differentchromosomes in the sample. The loci frequencies are summed for aparticular chromosome, and the sum of the loci are used to determineaneuploidy. This aspect of the invention sums the frequencies of theindividual loci on each chromosome and then compares the sum of the locion one chromosome against another chromosome to determine whether achromosomal abnormality exists.

The nucleic acids analyzed using the assay systems of the invention arepreferably selectively amplified and optionally isolated from the mixedsample using primers specific to the locus of interest (e.g., to a locusof interest in a mixed sample). The primers for such selectiveamplification designed to isolate regions may be chosen for variousreasons, but are preferably designed to 1) efficiently amplify a regionfrom the chromosome of interest; 2) have a predictable range ofexpression from maternal and/or fetal sources in different mixedsamples; 3) be distinctive to the particular chromosome, i.e., notamplify homologous regions on other chromosomes. The following areexemplary techniques that may be employed in the assay system or theinvention.

The assay system of the invention detects both fetal aneuploidies andspecific chromosomal abnormalities through identification andquantification of specific loci of interest. Such additionalabnormalities include, but are not limited to, deletion mutations,insertion mutations, copy number polymorphisms, copy number variants,chromosome 22q11 deletion syndrome, 11q deletion syndrome on chromosome11, 8p deletion syndrome on chromosome 8, and the like. Generally, atleast two selected nucleic acid sequences present on the same orseparate chromosomes are analyzed, and at least one of the selected lociis associated with the fetal allelic abnormality. The sequences of thetwo selected loci and number of copies of the two selected loci are thencompared to determine whether the chromosomal abnormality is present,and if so, the nature of the abnormality.

While much of the description contained herein describes detectinganeuploidy by counting the abundance of loci on one or more putativeaneuploid chromosomes and the abundance of loci on one or more normalchromosomes, the same techniques may be used to detect copy numbervariations where such copy number variation occurs on only a portion ofa chromosome. In this detection of the copy number variations, multipleloci within the putative copy number variation location are compared tomultiple loci outside of the putative copy number variation location.For instance, one may detect a chromosome 22q11 deletion syndrome in afetus in a maternal sample by selecting two or more nucleic regionswithin the 22q11 deletion and two or more loci outside of the 22q11deletion. The loci outside of the 22q11 deletion may be on anotherregion of Chromosome 22 or may be on a completely different chromosome.The abundance of each loci is determined by the methods described inthis application.

In some aspects a universal amplification may be used for amplifying theloci. In some aspects, the loci for each sample are assayed in a singlereaction in a single vessel. In other aspects, loci from multiplesamples can be assayed in a single reaction in a single vessel.

Certain aspects of the invention can detect a deletion, including theboundaries of such deletions. In some aspects, at least 24 selected locimay be used within the region of the putative deletion and at least 24selected loci may be used outside of the region of the putativedeletion. In another aspect at least 48 selected loci may be used withinthe region of the putative deletion and at least 48 selected loci may beused outside of the region of the putative deletion. In another aspectat least 48 selected loci may be used within the region of the putativedeletion and at least 96 selected loci may be used outside of the regionof the putative deletion. In another aspect at least 48 selected locimay be used within the region of the putative deletion and at least 192selected loci may be used outside of the region of the putativedeletion. In a preferred aspect at least 384 selected loci may be usedwithin the region of the putative deletion and at least 384 selectedloci may be used outside of the region of the putative deletion. Theloci within the deletion are then summed as are the loci outside of thedeletion. These sums are then compared to each other to determine thepresence or absence of a deletion. Optionally, the sums are put into aratio and that ratio may be compared to an average ratio created from anormal population. When the ratio for a sample falls statisticallyoutside of an expected ratio, the deletion is detected. The thresholdfor the detection of a deletion may be twice or more, preferably four ormore times the variation calculated in the normal population.

Polymorphisms Associated with Diseases or Predispositions

The assay systems of the invention are utilized to detect polymorphisms,such as those associated with an autosomal dominant or recessive diseaseor predisposition disorder. Given the multiplexed nature of the assaysystems of the invention, this detection takes place in the same assayas the detection of chromosomal abnormalities. Thus a single assaysystem can provide diagnostic information on different classes ofgenetic mutations. Accordingly, as the preferred assay systems of theinvention are highly multiplexed and able to interrogate hundreds oreven thousands of nucleic acids within a mixed sample, in certainaspects it is desirable to interrogate the sample for nucleic acidmarkers within the mixed sample, e.g., nucleic acids associated withgenetic risk or that identify the presence or absence of infectiousorganisms. Thus, the assay systems provide detection of such nucleicacids in conjunction with the detection of nucleic acids for copy numberdetermination within a mixed sample.

Thus, the assay system of the invention can be used to detectpolymorphisms in a mixed sample, where such polymorphisms are associatedwith genes associated with autosomal recessive disorders, mutationsassociated with autosomal dominant disorders; polymorphisms associatedwith risk of developing a disease and/or disease progression (e.g.,metastasis) and prognosis indicators.

In other specific aspects, the assay system of the invention can be usedto detect fetal mutations or polymorphisms in a maternal sample, wheresuch mutations or polymorphisms are associated with polygenic disorderssuch as coronary heart disease, diabetes, hypertension, congenital heartdefects, and epilepsy. Examples include mutations in genes associatedwith predispositions such as mutations in cancer susceptibility genes,(e.g. mutations in BRCAI or II or in p53); polymorphisms associated withincreased risk of developing later onset diseases, such as the apoE3gene polymorphism associated with Alzheimer's risk,

In addition to detection of chromosomal abnormalities and single genemutations or polymorphisms associated with monogenic or polygenicdisease, disorders or predispositions, the assay systems of theinvention may identify infectious agents in the mixed sample.

Selected Amplification

Numerous selective amplification methods can be used to provide theamplified nucleic acids that are analyzed in the assay systems of theinvention, and such methods are preferably used to increase the copynumbers of a locus of interest in a mixed sample in a manner that allowspreservation of information concerning the initial content of the locusin the mixed sample. Although not all combinations of amplification andanalysis are described herein in detail, it is well within the skill ofthose in the art to utilize different amplification methods and/oranalytic tools to isolate and/or analyze the nucleic acids of regionconsistent with this specification, and such variations will be apparentto one skilled in the art upon reading the present disclosure.

Such amplification methods include but are not limited to, polymerasechain reaction (PCR) (U.S. Pat. Nos. 4,683,195; and 4,683,202; PCRTechnology: Principles and Applications for DNA Amplification, ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992), ligase chain reaction (LCR) (Wuand Wallace, Genomics 4:560, 1989; Landegren et al., Science 241:1077,1988), strand displacement amplification (SDA) (U.S. Pat. Nos.5,270,184; and 5,422,252), transcription-mediated amplification (TMA)(U.S. Pat. No. 5,399,491), linked linear amplification (LLA) (U.S. Pat.No. 6,027,923), and the like, self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NASBA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be usedinclude: Qbeta Replicase, described in PCT Patent Application No.PCT/US87/00880, isothermal amplification methods such as SDA, describedin Walker et al., Nucleic Acids Res. 20(7):1691-6 (1992), and rollingcircle amplification, described in U.S. Pat. No. 5,648,245. Otheramplification methods that may be used are described in, U.S. Pat. Nos.5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317 and USPub. No. 20030143599, each of which is incorporated herein by reference.In some aspects DNA is amplified by multiplex locus-specific PCR. In apreferred aspect the DNA is amplified using adaptor-ligation and singleprimer PCR. Other available methods of amplification, such as balancedPCR (Makrigiorgos, et al., Nature Biotechnol, 20:936-9 (2002)) andisothermal amplification methods such as nucleic acid sequence basedamplification (NASBA) and self-sustained sequence replication (Guatelliet al., PNAS USA 87:1874 (1990)). Based on such methodologies, a personskilled in the art readily can design primers in any suitable regions 5′and 3′ to a locus of interest. Such primers may be used to amplify DNAof any length so long that it contains the locus of interest in itssequence.

The length of an amplified selected locus from a genomic region ofinterest is long enough to provide enough sequence information todistinguish it from other nucleic acids that are amplified and/orselected. Generally, an amplified nucleic acid is at least about 16nucleotides in length, and more typically, an amplified nucleic acid isat least about 20 nucleotides in length. In a preferred aspect of theinvention, an amplified nucleic acid is at least about 30 nucleotides inlength. In a more preferred aspect of the invention, an amplifiednucleic acid is at least about 32, 40, 45, 50, or 60 nucleotides inlength. In other aspects of the invention, an amplified nucleic acid canbe about 100, 150 or up to 200 in length.

In certain aspects, the selected amplification comprises an initiallinear amplification step. This can be particularly useful if thestarting amount of DNA from the mixed sample is quite limited, e.g.,where the cell-free DNA in a sample is available in limited quantities.This mechanism increases the amount of DNA molecules that arerepresentative of the original DNA content, and help to reduce samplingerror where accurate quantification of the DNA or a fraction of the DNA(e.g., fetal DNA contribution in a maternal sample) is needed.

Thus, in one aspect, a limited number of cycles of sequence-specificlinear amplification are performed on the starting mixed samplecomprising cfDNA. The number of cycles is generally less than that usedfor a typical PCR amplification, e.g., 5-30 cycles or fewer. Primers orprobes may be designed to amplify specific genomic segments or regions.The primers or probes may be modified with an end label at the 5′ end(e.g. with biotin) or elsewhere along the primer or probe such that theamplification products could be purified or attached to a solidsubstrate (e.g., bead or array) for further isolation or analysis. In apreferred aspect, the primers are multiplexed such that a singlereaction yields multiple DNA fragments from different regions.Amplification products from the linear amplification could then befurther amplified with standard PCR methods or with additional linearamplification.

For example, cfDNA can be isolated from blood, plasma, or serum from apregnant woman, and incubated with primers against a set number of locithat correspond to chromosomes of interest. Preferably, the number ofprimers used for initial linear amplification will be 12 or more, morepreferably 24 or more, more preferably 36 or more, even more preferably48 or more, and even more preferably 96 or more. Each of the primerscorresponds to a single locus, and is optionally tagged foridentification and/or isolation. A limited number of cycles, preferably10 or fewer, are performed with linear amplification. The amplificationproducts are subsequently isolated, e.g., when the primers are linked toa biotin molecule the amplification products can be isolated via bindingto avidin or streptavidin on a solid substrate. The products are thensubjected to further biochemical processes such as further amplificationwith other primers and/or detection techniques such as sequencedetermination and hybridization.

Efficiencies of linear amplification may vary between sites and betweencycles so that in certain systems normalization may be used to ensurethat the products from the linear amplification are representative ofthe nucleic acid content starting material. One practicing the assaysystem of the invention can utilize information from various samples todetermine variation in nucleic acid levels, including variation indifferent loci in individual samples and/or between the same loci indifferent samples following the limited initial linear amplification.Such information can be used in normalization to prevent skewing ofinitial levels of DNA content.

Universal Amplification

In preferred aspects of the invention, the selectively amplified lociare preferably further amplified through universal amplification of allor substantially all of the various loci to be analyzed using the assaysystems of the invention. Universal primer regions are added to thefixed sequence oligonucleotides so that the selectively amplified locimay be further amplified in a single universal amplification reaction.These universal primer sequences may be added to the nucleic acidsregions during the selective amplification process, i.e., the primersfor selective amplification have universal primer sequences that flank alocus. Alternatively, adapters comprising universal amplificationsequences can be added to the ends of the selected nucleic acids asadapters following amplification and isolation of the selected nucleicacids from the mixed sample.

In one exemplary aspect, nucleic acids are initially amplified from amixed sample using primers complementary to selected regions of thechromosomes of interest, followed by a universal amplification step toincrease the number of loci for analysis. This introduction of primerregions to the initial amplification products from a mixed sample allowsa subsequent controlled universal amplification of all or a portion ofselected nucleic acids prior to or during analysis, e.g. sequencedetermination.

Bias and variability can be introduced during DNA amplification, such asthat seen during polymerase chain reaction (PCR). In cases where anamplification reaction is multiplexed, there is the potential that lociwill amplify at different rates or efficiency. Part of this may be dueto the variety of primers in a multiplex reaction with some havingbetter efficiency (i.e. hybridization) than others, or some workingbetter in specific experimental conditions due to the base composition.Each set of primers for a given locus may behave differently based onsequence context of the primer and template DNA, buffer conditions, andother conditions. A universal DNA amplification for a multiplexed assaysystem will generally introduce less bias and variability.

Accordingly, in a one aspect, a small number (e.g., 1-10, preferably3-5) of cycles of selected amplification or nucleic acid enrichment ofthe initial sample in a multiplexed mixture reaction are performed,followed by universal amplification using introduced universal primers.The number of cycles using universal primers will vary, but willpreferably be at least 10 cycles, more preferably at least 5 cycles,even more preferably 20 cycles or more. By moving to universalamplification following a lower number of amplification cycles, the biasof having certain loci amplify at greater rates than others is reduced.

Optionally, the assay system will include a step between the selectedamplification and universal amplification to remove any excess nucleicacids that are not specifically amplified in the selected amplification.

The whole product or an aliquot of the product from the selectedamplification may be used for the universal amplification. The same ordifferent conditions (e.g., polymerase, buffers, and the like) may beused in the amplification steps, e.g., to ensure that bias andvariability are not inadvertently introduced due to experimentalconditions. In addition, variations in primer concentrations may be usedto effectively limit the number of sequence specific amplificationcycles.

In certain aspects, the universal primer regions of the primers oradapters used in the assay system are designed to be compatible withconventional multiplexed assay methods that utilize general primingmechanisms to analyze large numbers of nucleic acids simultaneously inone reaction in one vessel. Such “universal” priming methods allow forefficient, high volume analysis of the quantity of loci present in amixed sample, and allow for comprehensive quantification of the presenceof loci within such a mixed sample for the determination of aneuploidy.

Examples of such assay methods include, but are not limited to,multiplexing methods used to amplify and/or genotype a variety ofsamples simultaneously, such as those described in Oliphant et al., U.S.Pat. No. 7,582,420.

Some aspects utilize coupled reactions for multiplex detection ofnucleic acid sequences where oligonucleotides from an early phase ofeach process contain sequences which may be used by oligonucleotidesfrom a later phase of the process. Exemplary processes for amplifyingand/or detecting nucleic acids in samples can be used, alone or incombination, including but not limited to the methods described below,each of which are incorporated by reference in their entirety.

In certain aspects, the assay system of the invention utilizes one ofthe following combined selective and universal amplification techniques:(1) ligase detection reaction (“LDR”) coupled with polymerase chainreaction (“PCR”); (2) primary PCR coupled to secondary PCR coupled toLDR; and (3) primary PCR coupled to secondary PCR. Each of these aspectsof the invention has particular applicability in detecting certainnucleic acid characteristics. However, each requires the use of coupledreactions for multiplex detection of nucleic acid sequence differenceswhere oligonucleotides from an early phase of each process containsequences which may be used by oligonucleotides from a later phase ofthe process.

Barany et al., U.S. Pat. Nos. 6,852,487, 6,797,470, 6,576,453,6,534,293, 6,506,594, 6,312,892, 6,268,148, 6,054,564, 6,027,889,5,830,711, 5,494,810, describe the use of the ligase chain reaction(LCR) assay for the detection of specific sequences of nucleotides in avariety of nucleic acid samples.

Barany et al., U.S. Pat. Nos. 7,807,431, 7,455,965, 7,429,453,7,364,858, 7,358,048, 7,332,285, 7,320,865, 7,312,039, 7,244,831,7,198,894, 7,166,434, 7,097,980, 7,083,917, 7,014,994, 6,949,370,6,852,487, 6,797,470, 6,576,453, 6,534,293, 6,506,594, 6,312,892, and6,268,148 describe LDR coupled PCR for nucleic acid detection.

Barany et al., U.S. Pat. Nos. 7,556,924 and 6,858,412, describe the useof precircle probes (also called “padlock probes” or “multi-inversionprobes”) with coupled LDR and polymerase chain reaction (“PCR”) fornucleic acid detection.

Barany et al., U.S. Pat. Nos. 7,807,431, 7,709,201, and 7,198, 814describe the use of combined endonuclease cleavage and ligationreactions for the detection of nucleic acid sequences.

Willis et al., U.S. Pat. Nos. 7,700,323 and 6,858,412, describe the useof precircle probes in multiplexed nucleic acid amplification, detectionand genotyping.

Ronaghi et al., U.S. Pat. No. 7,622,281 describes amplificationtechniques for labeling and amplifying a nucleic acid using an adaptercomprising a unique primer and a barcode.

In some cases, a single assay may employ a combination of theabove-described methods. For example, some of the loci may be detectedusing fixed sequence oligonucleotides that bind to adjacent,complementary regions on a locus, while other loci may be detected usingbridging loci in the same assay. In another example, some of the locimay be detected using fixed sequence oligonucleotides that bind toadjacent, complementary regions on a locus, while other loci may requirea primer extension step to join the fixed sequence oligonucleotides.

In a preferred aspect, the amplification products are multiplexed, asdescribed previously. In a preferred aspect, the multiplex amplificationproducts are quantified by analysis of the amplification products. In apreferred aspect, a representational sample of individual molecules fromthe amplification processes is isolated from the other molecules forfurther analysis. To obtain a representational sample of individualmolecules, the average number of molecules per locus must exceed thesampling noise created by the multiplexed reaction. In one aspect, theaverage number per locus is greater than 100. In another aspect, theaverage number per locus is greater than 500. In another aspect theaverage number per locus is greater than 1000.

Individual molecules from the amplification product are preferablyisolated physically from the other molecules in a manner that allows thedifferent amplification products to be distinguished from one another inanalysis. In a preferred aspect, this isolation occurs on a solidsubstrate. The isolated molecule may be associated with a particularidentifiable or physical address either prior to analysis, or theaddress may become known for the particular amplification products basedon the outcome of the analysis. The substrate may be a planar surface orthree-dimensional surface such as a bead.

Once isolated, the individual amplification product may be furtheramplified to make multiple identical copies of that molecule at the sameknown or identifiable location. The amplification may occur before orafter that location becomes an identifiable or physical address. Theamplification product and or its copies (which may be identical orcomplementary to the amplification product) are then analyzed based onthe sequence of the amplification product or its copies to identify theparticular locus and/or allele it represents.

In a preferred aspect, the entire length of the amplification product ora portion of the amplification product may be analyzed using sequencedetermination. The number of bases that need to be determined must besufficient to uniquely identify the amplification product as belongingto a specific locus and/or allele. In one preferred aspect, theamplification product is analyzed through sequence determination of theselected amplification product.

Numerous methods of sequence determination are compatible with the assaysystems of the inventions. Exemplary methods for sequence determinationinclude, but are not limited to, including, but not limited to,hybridization-based methods, such as disclosed in Drmanac, U.S. Pat.Nos. 6,864,052; 6,309,824; and 6,401,267; and Drmanac et al, U.S. patentpublication 2005/0191656, which are incorporated by reference,sequencing by synthesis methods, e.g., Nyren et al, U.S. Pat. Nos.7,648,824, 7,459,311 and 6,210,891; Balasubramanian, U.S. Pat. Nos.7,232,656 and 6,833,246; Quake, U.S. Pat. No. 6,911,345; Li et al, Proc.Natl. Acad. Sci., 100: 414-419 (2003); pyrophosphate sequencing asdescribed in Ronaghi et al., U.S. Pat. Nos. 7,648,824, 7,459,311,6,828,100, and 6,210,891; and ligation-based sequencing determinationmethods, e.g., Drmanac et al., U.S. Pat. Appln No. 20100105052, andChurch et al, U.S. Pat. Appln Nos. 20070207482 and 20090018024.

Sequence information may be determined using methods that determine many(typically thousands to billions) of nucleic acid sequences in anintrinsically parallel manner, where many sequences are read outpreferably in parallel using a high throughput serial process. Suchmethods include but are not limited to pyrosequencing (for example, ascommercialized by 454 Life Sciences, Inc., Branford, Conn.); sequencingby ligation (for example, as commercialized in the SOLiD™ technology,Life Technology, Inc., Carlsbad, Calif.); sequencing by synthesis usingmodified nucleotides (such as commercialized in TruSeq™ and HiSeq™technology by Illumina, Inc., San Diego, Calif., HELISCOPE′ by HelicosBiosciences Corporation, Cambridge, Mass., and PacBio RS by PacificBiosciences of California, Inc., Menlo Park, Calif.), sequencing by iondetection technologies (Ion Torrent, Inc., South San Francisco, Calif.);sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View,Calif.); nanopore-based sequencing technologies (for example, asdeveloped by Oxford Nanopore Technologies, LTD, Oxford, UK), and likehighly parallelized sequencing methods.

Alternatively, in another aspect, the entire length of the amplificationproduct or a portion of the amplification product may be analyzed usinghybridization techniques. Methods for conducting polynucleotidehybridization assays for detection of have been well developed in theart. Hybridization assay procedures and conditions will vary dependingon the application and are selected in accordance with the generalbinding methods known including those referred to in: Maniatis et al.Molecular Cloning: A Laboratory Manual (2nd Ed. Cold Spring Harbor,N.Y., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide toMolecular Cloning Techniques (Academic Press, Inc., San Diego, Calif.,1987); Young and Davis, P.N.A.S, 80: 1194 (1983). Methods and apparatusfor carrying out repeated and controlled hybridization reactions havebeen described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and6,386,749, 6,391,623 each of which are incorporated herein by reference.

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred aspects. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Patent application 60/364,731 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application60/364,731 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

Variation Minimization within and Between Samples

One challenge with the detection of chromosomal abnormalities in a fetusby detection in a mixed sample is that the nucleic acids from the minorsource are present in much lower abundance than the nucleic acids fromnormal cell type.

The variation between levels found between samples and/or for lociwithin a sample may be minimized in a combination of analytical methods,many of which are described in this application. For instance, variationis lessened by using an internal reference in the assay. An example ofan internal reference is the use of a chromosome present in a “normal”abundance (e.g., disomy for an autosome) to compare against a chromosomepresent in putatively abnormal abundance, such as aneuploidy, in thesame sample. While the use of one such “normal” chromosome as areference chromosome may be sufficient, it is also possible to use twoor more normal chromosomes as the internal reference chromosomes toincrease the statistical power of the quantification.

One method of using an internal reference is to calculate a ratio ofabundance of the putatively abnormal chromosomes to the abundance of thenormal chromosomes in a sample, called a chromosomal ratio. Incalculating the chromosomal ratio, the abundance or counts of each ofthe loci for each chromosome are summed together to calculate the totalcounts for each chromosome. The total counts for one chromosome are thendivided by the total counts for a different chromosome to create achromosomal ratio for those two chromosomes.

Alternatively, a chromosomal ratio for each chromosome may be calculatedby first summing the counts of each of the loci for each chromosome, andthen dividing the sum for one chromosome by the total sum for two ormore chromosomes. Once calculated, the chromosomal ratio is thencompared to the average chromosomal ratio from a normal population.

The average may be the mean, median, mode or other average, with orwithout normalization and exclusion of outlier data. In a preferredaspect, the mean is used. In developing the data set for the chromosomalratio from the normal population, the normal variation of the measuredchromosomes is calculated. This variation may be expressed a number ofways, most typically as the coefficient of variation, or CV. When thechromosomal ratio from the sample is compared to the average chromosomalratio from a normal population, if the chromosomal ratio for the samplefalls statistically outside of the average chromosomal ratio for thenormal population, the sample contains an aneuploidy. The criteria forsetting the statistical threshold to declare an aneuploidy depend uponthe variation in the measurement of the chromosomal ratio and theacceptable false positive and false negative rates for the desiredassay. In general, this threshold may be a multiple of the variationobserved in the chromosomal ratio. In one example, this threshold isthree or more times the variation of the chromosomal ratio. In anotherexample, it is four or more times the variation of the chromosomalratio. In another example it is five or more times the variation of thechromosomal ratio. In another example it is six or more times thevariation of the chromosomal ratio. In the example above, thechromosomal ratio is determined by summing the counts of loci bychromosome. Typically, the same number of selected loci for eachchromosome is used. An alternative method for generating the chromosomalratio would be to calculate the average counts for the loci for eachchromosome. The average may be any estimate of the mean, median or mode,although typically an average is used. The average may be the mean ofall counts or some variation such as a trimmed or weighted average. Oncethe average counts for each chromosome have been calculated, the averagecounts for each chromosome may be divided by the other to obtain achromosomal ratio between two chromosomes, the average counts for eachchromosome may be divided by the sum of the averages for all measuredchromosomes to obtain a chromosomal ratio for each chromosome asdescribed above. As highlighted above, the ability to detect ananeuploidy in a maternal sample where the putative DNA is in lowrelative abundance depends greatly on the variation in the measurementsof different selected loci in the assay. Numerous analytical methods canbe used which reduce this variation and thus improve the sensitivity ofthis method to detect aneuploidy. One method for reducing variability ofthe assay is to increase the number of selected loci used to calculatethe abundance of the chromosomes. In general, if the measured variationof a single selected locus of a chromosome is X % and Y differentselected loci are measured on the same chromosome, the variation of themeasurement of the chromosomal abundance calculated by summing oraveraging the abundance of each selected locus on that chromosome willbe approximately X % divided by Y^½. Stated differently, the variationof the measurement of the chromosome abundance would be approximatelythe average variation of the measurement of each selected locus'abundance divided by the square root of the number of loci.

In a preferred aspect of this invention, the number of loci measured foreach chromosome is at least 24. In another preferred aspect of thisinvention, the number of selected loci measured for each chromosome isat least 48. In another preferred aspect of this invention, the numberof selected loci measured for each chromosome is at least 100. Inanother preferred aspect of this invention the number of selected locimeasured for each chromosome is at least 200. There is incremental costto measuring each locus and thus it is important to minimize the numberof each selected locus. In a preferred aspect of this invention, thenumber of selected loci measured for each chromosome is less than 2000.In a preferred aspect of this invention, the number of selected locimeasured for each chromosome is less than 1000. In a most preferredaspect of this invention, the number of selected loci measured for eachchromosome is at least 48 and less than 1000. In one aspect, followingthe measurement of abundance for each selected locus, a subset of theselected loci may be used to determine the presence or absence ofaneuploidy. There are many standard methods for choosing the subset ofselected loci. These methods include outlier exclusion, where theselected loci with detected levels below and/or above a certainpercentile are discarded from the analysis. In one aspect, thepercentile may be the lowest and highest 5% as measured by abundance. Inanother aspect, the percentile may be the lowest and highest 10% asmeasured by abundance. In another aspect, the percentile may be thelowest and highest 25% as measured by abundance.

Another method for choosing the subset of selected loci includes theelimination of regions that fall outside of some statistical limit. Forinstance, selected loci that fall outside of one or more standarddeviations of the mean abundance may be removed from the analysis.Another method for choosing the subset of selected loci may be tocompare the relative abundance of a selected locus to the expectedabundance of the same selected locus in a healthy population and discardany selected loci that fail the expectation test. To further minimizethe variation in the assay, the number of times each selected locus ismeasured may be increased. As discussed, in contrast to the randommethods of detecting aneuploidy where the genome is measured on averageless than once, the assay systems of the present invention intentionallymeasures each selected locus multiple times. In general, when countingevents, the variation in the counting is determined by Poissonstatistics, and the counting variation is typically equal to one dividedby the square root of the number of counts. In a preferred aspect of theinvention, the selected loci are each measured on average at least 100times. In a preferred aspect to the invention, the selected loci areeach measured on average at least 500 times. In a preferred aspect tothe invention, the selected loci are each measured on average at least1000 times. In a preferred aspect to the invention, the selected lociare each measured on average at least 2000 times. In a preferred aspectto the invention, the selected loci are each measured on average atleast 5000 times.

In another aspect, subsets of loci can be chosen randomly but withsufficient numbers of loci to yield a statistically significant resultin determining whether a chromosomal abnormality exists. Multipleanalyses of different subsets of loci can be performed within a mixedsample to yield more statistical power. In this example, it may or maynot be necessary to remove or eliminate any loci prior to the randomanalysis. For example, if there are 100 selected loci for chromosome 21and 100 selected loci for chromosome 18, a series of analyses could beperformed that evaluate fewer than 100 loci for each of the chromosomes.

In addition to the methods above for reducing variation in the assay,other analytical techniques, many of which are described earlier in thisapplication, may be used in combination. In general, the variation inthe assay may be reduced when all of the loci for each sample areinterrogated in a single reaction in a single vessel. Similarly, thevariation in the assay may be reduced when a universal amplificationsystem is used. Furthermore, the variation of the assay may be reducedwhen the number of cycles of amplification is limited.

Use of Assay Systems for Detection in Mixed Samples from Cancer Patients

The assay system allow the detection of quantitative and qualitativetumor-specific alterations of cfDNA, such as DNA strand integrity,frequency of mutations, abnormalities of microsatellites, andmethylation of genes, as diagnostic, prognostic, and monitoring markersin cancer patients. The ability to combine such detection of single genealterations (including point mutations, indels and copy numbervariation) with CNV detection provides a powerful methods for assistingwith clinical diagnosis, treatments, outcome prediction and progressionmonitoring in patients with or suspected of having a malignancy.

In some aspects, the assay system of the invention is used fordiagnostic purposes e.g., to detect the presence and/or nature of amalignancy in a patient or to provide a quantitative estimate of tumorload in a patient. Circulating tumor DNA and microRNAs have beenassociated with certain cancers, such as lung cancer (Roth C et al., MolOncol. 2011 June; 5(3):281-91. Epub 2011 Feb. 24). Copy numbervariations have also been detected in certain cancers, such as amplifiedHER2 and estrogen receptor in the cfDNA in breast cancer patients. (PageK., Br J Cancer. 2011 Apr. 12; 104(8):1342-8. Epub 2011 Mar. 22).

In other aspects of the invention, the assay system is used in cancerpatients to monitor a response to treatment and/or to follow progress ofthe disease, e.g., to measure single gene alterations and cfDNA inpatients receiving chemoradiotherapy (CRT). For certain cancers, it hasbeen shown that cfDNA integrity index can be significantly andindependently associated with tumor response to treatment. Agostini M etal., Ann Surg Oncol. 2011 Mar. 17. Also, the presence or absence ofcertain genetic alterations and/or differences in copy number variationhas been associated with response to chemotherapy and/or prognosis of adisease. See, e.g., Savas S., Acta Oncol. 2010 November; 49(8):1217-26.Epub 2010 Jul. 29, which describes useful genetic variations fordetermination of treatment response and survival in cancer. For example,the detection of cfDNA levels combined with detection of mutations inthe K-RAS gene and/or the p53 gene provide a powerful, relativelynon-invasive tool in measuring the prognosis of various cancers,including ovarian cancer, endometrial cancer and lymphomas. Dobrzycka Bet al., Ann Oncol. 2011 May; 22(5):1133-40. Epub 2010 Nov. 23; DobrzyckaB et al., Int J Cancer. 2010 Aug. 1; 127(3):612-21; Hosny G et al.,Cancer Lett. 2009 Mar. 18; 275(2):234-9. Epub 2008 Nov. 28. Suchanalysis can be further assisted using tools such as Varietas, afunctional database portal for identification of genetic variation andassociation with treatment outcomes and prognosis. Paananen J et al.,Database (Oxford). 2010 Jul. 29; 2010:baq016.

Use of Assay Systems for Detection of Mixed Samples from TransplantPatients

The assay systems of the invention can be used to monitor organ healthin a transplant patient using a combination of detection of cfDNA anddetection of SNPs or mutations in one or more single genes. Transplantedorgans have genomes that are distinct from the genome of a recipientpatient, and organ health can be detected using assay system. Forexample, acute cellular rejection has been shown to be associated withsignificantly increased levels of cell-free DNA from the donor genome inheart transplant recipients. Snyder T M et al., Proc Natl Acad Sci USA.2011 Apr. 12; 108(15):6229-34. Epub 2011 Mar. 28. In addition,chemokines and adhesion molecules mediate allograft rejection byrecruiting leukocytes into the allograft, and SNPs located ininterleukin (IL)-8, CXCR1, CXCR2, have been shown to correlate withallograft outcomes. Ro H. et al., Transplantation. 2011 Jan. 15;91(1):57-64. Thus, the assay systems of the invention can providenoninvasive tests for monitoring solid organ transplant recipients, andcan aid in identification of early signs of rejection without thenecessity of organ biopsies or other more onerous diagnostic orprognostic techniques.

Use of Assay Systems for Detection in Maternal Samples

In certain specific aspects, determining the relative percentage offetal DNA in a maternal sample may be beneficial in analyzing theamplification products, as percentage fetal DNA in the sample providesimportant information on the expected statistical presence ofchromosomes, and variation from that expectation may be indicative offetal aneuploidy. This may be especially helpful in circumstances wherethe level of fetal DNA in a maternal sample is low, as the percent fetalcontribution can be used in determining the quantitative statisticalsignificance in the variations of levels of identified selected loci ina maternal sample. In other aspects, the determining of the relativepercent fetal cfDNA in a maternal sample may be beneficial in estimatingthe level of certainty or power in detecting a fetal aneuploidy.

In some specific aspects, the relative fetal contribution of maternalDNA at the allele of interest can be compared to the paternalcontribution at that allele to determine approximate fetal DNAconcentration in the sample. In other specific aspects, the relativequantity of solely paternally-derived sequences (e.g., Y-chromosomesequences or paternally-specific polymorphisms) can be used to determinethe relative concentration of fetal DNA in a maternal sample.

Another exemplary approach to determining the percent fetal contributionin a maternal sample through the analysis of DNA fragments withdifferent patterns of DNA methylation between fetal and maternal DNA. Ina preferred aspect, the amplified DNA from plasma free DNA is bypolymerase chain reaction (PCR). Other mechanisms for amplification canbe used as well, including those described in more detail herein, aswill be apparent to one skilled in the art upon reading the presentdisclosure.

In particular aspects, the percentage of free fetal DNA in the maternalsample can determined by PCR using serially diluted DNA isolated fromthe maternal sample, which can accurately quantify the number of genomescomprising the amplified genes.

In circumstances where the fetus is male, percent fetal DNA in a samplecan be determined through detection of Y-specific loci and comparison tocalculated maternal DNA content. Quantities of an amplified Y-specificlocus, such as a region from the sex-determining region Y gene (SRY),which is located on the Y chromosome and is thus representative of fetalDNA, can be determined from the sample and compared to one or moreamplified selected loci which are present in both maternal DNA and fetalDNA and which are preferably not from a chromosome believed topotentially be aneuploid in the fetus, e.g., an autosomal region that isnot on chromosome 21 or 18. Preferably, this amplification step isperformed in parallel with the selective amplification step, although itmay be performed either before or after the selective amplificationdepending on the nature of the multiplexed assay.

In particular aspects, the percentage of cell-free fetal DNA in amaternal sample can determined by PCR using serially diluted DNAisolated from the maternal sample, which can accurately quantify thenumber of genomes comprising the amplified genes. PCR using seriallydiluted DNA isolated from the maternal sample may be preferred whendetermining percent fetal DNA with a male fetus. For example, if theblood sample contains 100% male fetal DNA, and 1:2 serial dilutions areperformed, then on average the Y-linked signal will disappear 1 dilutionbefore the autosomal signal, since there is 1 copy of the Y-linked geneand 2 copies of the autosomal gene.

In a specific aspect, the percentage of free fetal DNA in maternalplasma is calculated for a male fetus using the following formula:percentage of free fetal DNA=(No. of copies of Y-linked gene×2×100)/(No.of copies of autosomal gene), where the number of copies of each gene isdetermined by observing the highest serial dilution in which the genewas detected. The formula contains a multiplication factor of 2, whichis used to normalize for the fact that there is only 1 copy of theY-linked gene compared to two copies of the autosomal gene in eachgenome, fetal or maternal.

Determination of Minor Source DNA Content in a Mixed Sample

In certain aspects of the invention, determination of the contributionof DNA form a minor source may be useful in determining copy numbervariation of loci in those samples. For example, in eachmaternally-derived sample, the DNA from a fetus will have approximately50% of its loci inherited from the mother and 50% of the loci inheritedfrom the father. Determining the loci contributed to the fetus frompaternal sources can allow the estimation of fetal DNA in a maternalsample, and thus provide information used to calculate the statisticallysignificant differences in chromosomal frequencies for chromosomes ofinterest.

In certain aspects, the determination of minor source polymorphismsrequires targeted SNP and/or mutation analysis to identify the presenceof the minor source DNA in a mixed sample. In some aspects, the use ofprior genotyping is helpful, e.g., genotyping of the donor of atransplant, genotyping of the father and mother in a maternal sample.But generally this information pertaining to the prior genotyping is notnecessary prior to performing the assay, and preferably the genotypingis performed simultaneously with the determination of copy number ofselected loci within a mixed sample.

In one preferred aspect, the percent minor source nucleic acids in amixed sample can be quantified using multiplexed SNP detection withoutusing prior genotypic knowledge. In this aspect, two or more selectedpolymorphic loci with a known SNP in each region are used. In apreferred aspect, the selected polymorphic loci are lociamplified. In apreferred aspect, the amplification is universal. In a preferredembodiment, the selected polymorphic loci are amplified in one reactionin one vessel. Each allele of the selected polymorphic loci in thematernal sample is determined and quantified. In a preferred aspect,high throughput sequencing is used for such determination andquantification. Loci are identified where the major and minor sourcegenotypes are different, e.g., the donor genotype is homozygous and therecipient genotype is heterozygous. This identification is done byobserving a high relative frequency of one allele (>80%) and a lowrelative frequency (<20% and >0.15%) of the other allele for aparticular selected locus. The use of multiple loci is particularlyadvantageous as it reduces the amount of variation in the measurement ofthe abundance of the alleles. All or a subset of the loci that meet thisrequirement are used to determine minor source nucleic acidconcentration through statistical analysis. In one aspect, concentrationis determined by summing the low frequency alleles from two or more locitogether, dividing by the sum of the high frequency alleles andmultiplying by two. In another aspect, the percent minor source nucleicacid is determined by averaging the low frequency alleles from two ormore loci, dividing by the average of the high frequency alleles andmultiplying by two.

For many alleles, major and minor source nucleic acid sequences may behomozygous and identical, and as this information is not distinguishingit is not useful in the determination of minor source nucleic acid in amixed sample. The present invention utilizes allelic information wherethere is a distinguishable difference between the cell sources (e.g., afetal allele containing at least one allele that differs from thematernal allele) in calculations of minor source nucleic acidpercentages. Data pertaining to allelic regions that are the same forthe major and minor source are thus not selected for analysis, or areremoved from the pertinent data prior to determination of percentage soas not to swamp out the useful data.

Exemplary methods for quantifying fetal DNA in maternal plasma can befound, e.g., in Chu et al., Prenat Diagn 2010; 30:1226-1229, which isincorporated herein by reference.

In one aspect, selected loci may be excluded if the amount or frequencyof the region appears to be an outlier due to experimental error, orfrom idiopathic genetic bias within a particular sample. In anotheraspect, selected nucleic acids may undergo statistical or mathematicaladjustment such as normalization, standardization, clustering, ortransformation prior to summation or averaging. In another aspect,selected nucleic acids may undergo both normalization and dataexperimental error exclusion prior to summation or averaging.

In a preferred aspect, 12 or more loci are used for the analysis. Inanother preferred aspect, 24 or more loci are used for the analysis. Inanother preferred aspect, 48 or more loci are used for the analysis. Inanother aspect, one or more indices are used to identify the sample

In a specific aspect, minor source contribution can be quantified usingtandem SNP detection. Techniques for identifying tandem SNPs in DNAextracted from, e.g., a maternal sample are disclosed in Mitchell et al,U.S. Pat. No. 7,799,531 and U.S. patent application Ser. Nos.12/581,070, 12/581,083, 12/689,924, and 12/850,588. These describe thedifferentiation of fetal and maternal loci through detection of at leastone tandem single nucleotide polymorphism (SNP) in a maternal samplethat has a different haplotype between the fetal and maternal genome.Identification and quantification of these haplotypes can be performeddirectly on the maternal sample, as described in the Mitchell et al.disclosures, and used to determine the percent fetal contribution in thematernal sample.

Once the percent cfDNA has been calculated for the minor source, thisdata may be combined with methods for aneuploidy detection to determinethe likelihood that a mixed sample may contain an aneuploidy. In oneaspect, an aneuploidy detection methods that utilizes analysis of randomDNA segments is used, such as that described in, e.g., Quake, U.S.patent application Ser. No. 11/701,686; Shoemaker et al., U.S. patentapplication Ser. No. 12/230,628. In a preferred aspect, aneuploidydetection methods that utilize analysis of selected loci in a mixedsample include both regions for determination of minor source DNAcontent as well as non-polymorphic regions from two or more chromosomesto detect a chromosomal abnormality in a single reaction. The singlereaction helps to minimize the risk of contamination or bias that may beintroduced during various steps in the assay system which may otherwiseskew results when utilizing minor source DNA content to help determinethe presence or absence of a chromosomal abnormality. In other aspects,a selected locus or regions may be utilized both for determination ofminor source DNA content as well as detection of minor sourcechromosomal abnormalities. Utilizing the same regions for both DNAcontent and detection of chromosomal abnormalities may further helpminimize any bias due to experimental error or contamination.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees centigrade, and pressure is at or nearatmospheric.

Example 1: General Aspects of the Assay Systems of the Invention

A number of assay formats were tested to demonstrate the ability toperform selective amplification and detection of independent loci todemonstrate multiplexed, ligation-based detection of a large number(e.g., 96 or more) of loci of interest. These loci included loci thatwere indicative of the presence of a particular chromosome or thepresence or absence of a mutation or polymorphism in a particularallele.

These assays were designed based on human genomic sequences, and eachinterrogation consisted of two fixed sequence oligos per selected locusinterrogated in the assay. The first oligo, complementary to the 3′region of a genomic region, comprised the following sequential (5′ to3′) oligo elements: a universal PCR priming sequence common to allassays: TACACCGGCGTTATGCGTCGAGAC (SEQ ID NO:1); a nine nucleotideidentification index specific to the selected locus; a 9 base locus- orlocus/allele-specific sequence that acts as a locus index in the firstSNP-independent set and a locus/allele index in thepolymorphism-specific second set; a hybridization breaking nucleotidewhich is different from the corresponding base in the genomic locus; anda 20-24 bp sequence complementary to the selected genomic locus. Incases where a SNP or mutation was detected in this portion of theselected genomic locus, the allele-specific interrogation set consistedof two first fixed sequence tandem ligation primers, each with adifferent locus/allele index and a different allele-specific base at theSNP position. These first oligos were designed for each selected nucleicacid to provide a predicted uniform T_(m) with a two degree variationacross all interrogations in the assay set.

The second fixed sequence oligo, complementary to the 5′ region of thegenomic loci, comprised the following sequential (5′ to 3′) elements: a20-24b sequence complimentary to the 5′ region in the genomic locus; ahybridization breaking nucleotide different from the corresponding basein the genomic locus; and a universal PCR priming sequence which wascommon to all third oligos in the assay set: ATTGCGGGGACCGATGATCGCGTC(SEQ ID NO:2).

In cases where a SNP or mutation was detected in the selected genomiclocus, the allele-specific interrogation set consisted of two tandemligation primers, each with a different locus/allele index and adifferent allele-specific base at the mutation/SNP position. This secondfixed sequence oligo was designed for each selected nucleic acid toprovide a predicted uniform T_(m) with a two degree variation across allinterrogations in the assay set that was substantially the same T_(m)range as the first oligo set.

In certain tested aspects, one or more bridging oligos were used thatwere complementary to the genomic locus sequence between the regioncomplementary to the first and second fixed sequence oligos used foreach selected locus. In specific aspects tested, more than one bridgingoligo was used to span the gap between the fixed sequenceoligonucleotides, and the one or more bridging oligo may optionally bedesigned to identify one or more mutations or SNPs in the sequence. Thelength of the bridging oligonucleotides used in the assay systems variedfrom 5 to 36 base pairs.

All oligonucleotides used in the tandem ligation formats weresynthesized using conventional solid-phase chemistry. The second fixedsequence oligos and the bridging oligonucleotides were synthesized with5′ phosphate moieties to enable ligation to 3′ hydroxyl termini ofadjacent oligonucleotides.

Example 2: Preparation of DNA for Use in Tandem Ligation Procedures

Genomic DNA from a Caucasian male (NA12801) or a Caucasian female(NA11995) was obtained from Coriell Cell Repositories (Camden, N.J.) andfragmented by acoustic shearing (Covaris, Woburn, Mass.) to a meanfragment size of approximately 200 bp.

The Coriell DNA was biotinylated using standard procedures. Briefly, theCovaris fragmented DNA was end-repaired by generating the followingreaction in a 1.5 ml microtube: 5 μg DNA, 12 μl 10× T4 ligase buffer(Enzymatics, Beverly Mass.), 50 U T4 polynucleotide kinase (Enzymatics,Beverly Mass.), and H₂O to 120 μl. This was incubated at 37° C. for 30minutes. The DNA was diluted using 10 mM Tris 1 mM EDTA pH 8.5 todesired final concentration of ˜2 ng/μl.

5 μl DNA was placed in each well of a 96-well plate, and the platesealed with an adhesive plate sealer and spun for 10 seconds at 250×g.The plate was then incubated at 95° C. for 3 minutes, cooled to 25° C.,and spun again for 10 seconds at 250×g. A biotinylation master mix wasprepared in a 1.5 ml microtube to final concentration of: 1× TdT buffer(Enzymatics, Beverly, Mass.), 8U TdT (Enzymatics, Beverly, Mass.), 250μM CoCl₂, 0.01 nmol/μl biotin-16-dUTP (Roche, Nutley N.J.), and H₂O to1.5 ml. 15 μl of the master mix was aliquoted into each well of a 96well plate, and the plate sealed with adhesive plate sealer. The platewas spun for 10 seconds at 250×g and incubated for 37° C. for 60minutes. Following incubation, the plate was spun again for 10 secondsat 250×g, and 7.5 μl precipitation mix (1 μg/μl Dextran Blue, 3 mMNaOAC) was added to each well.

The plate was sealed with an adhesive plate sealer and mixed using anIKA plate vortexer for 2 minutes at 3000 rpm. 27.5 μl of isopropanol wasadded into each well, the plate sealed with adhesive plate sealer, andvortexed for 5 minutes at 3000 rpm. The plate was spun for 20 minutes at3000×g, the supernatant was decanted, and the plate inverted andcentrifuged at 10×g for 1 minute onto an absorbent wipe. The plate wasair-dried for 5 minutes, and the pellet resuspended in 30 μl 10 mM TrispH8.0, 1 mM EDTA.

Example 3: Exemplary Assay Formats Using Tandem Ligation

Numerous tandem ligation assay formats using the biotinylated DNA weretested to illustrate proof of concept for the assay systems of theinvention, and demonstrated the ability to perform highly multiplexed,targeted detection of a large number of independent loci using theseries of different assay formats. The exemplary assay systems of theinvention were designed to comprise 96 or more interrogations per lociin a genetic sample, and in cases where SNPs were detected the assayformats utilized 192 or more separate interrogations, each utilizing thedetection of different alleles per 96 loci in genetic samples. Theexamples described for each assay format utilized two different sets offixed sequence oligonucleotides and/or bridging oligos (as described inExample 1), comprising a total 96 or 192 interrogation reactions for theselected loci depending upon whether or not SNPs were identified.

A first exemplary assay format used locus-specific fixed sequence oligosand bridging oligos, where there was a one base gap between the firstfixed sequence oligo and the bridging oligos, and a second one base gapbetween the bridging oligos and the second fixed sequence oligo. Each ofthe two gaps encompassed two different SNPs. In this format, a DNApolymerase was used to incorporate each of the SNP bases, and ligase wasused to seal the nicks formed thereby. SNP base discrimination derivedfrom the fidelity of base incorporation by the polymerase, and in theevent of mis-incorporation, the tendency of ligase to not seal nicksadjacent to mismatched bases.

The second exemplary assay format used two locus-specific fixed sequenceoligonucleotides without a bridging oligo, where there was a ˜15-35 basegap between the fixed sequence oligos, and where the gap spanned one ormore SNPs. In this format, a polymerase was used to incorporate themissing bases of the gap, and a ligase was used to seal the nick formedthereby. SNP base discrimination derived from the fidelity of baseincorporation by the polymerase, and in the event of misincorporation,the tendency of ligase to not seal nicks adjacent to mismatched bases.

A third exemplary assay format used allele-specific first and secondfixed sequence oligos without a bridging oligo, where there was a ˜15-35base gap between the first and second fixed sequence oligos, and wherethe gap spanned one or more SNPs. Two separate allele-specific firstfixed sequence oligos and two separate allele-specific second fixedsequence oligos were used. A polymerase was used to incorporate themissing bases, and a ligase was used to seal the nick formed thereby.SNP base discrimination derived from hybridization specificity, thetendency of non-proofreading polymerase to not extend annealed primerswith mismatches near the 3′ end, and the tendency of the ligase to notseal nicks adjacent to mismatched bases.

A fourth exemplary format used allele-specific fixed sequence oligos anda locus-specific bridging oligo. In this format, two separate fixedsequence oligos complementary to the 3′ end of the loci of interest, thefirst with a 3′ base specific for one allele of the targeted SNP, andthe second with a 3′ base specific for the other allele of the targetedSNP. Similarly, two separate second fixed sequence oligos were used, thefirst with a 5′ base specific for one allele of a second targeted SNP,and the second with a 5′ base specific for the other allele of thesecond targeted SNP. The bridging oligos were complementary to theregion directly adjacent to the locus regions complementary to the firstand second fixed sequence oligos, and thus no polymerase was neededprior to ligation. Ligase was used to seal the nicks between the fixedsequence oligos and the bridging oligo. SNP base discrimination in thisassay format derived from hybridization specificity and the tendency ofthe ligase to not seal nicks adjacent to mismatched bases. Thisexemplary format was tested using either T4 ligase or Taq ligase forcreation of the contiguous template, and both were proved effective inthe reaction as described below.

A fifth exemplary format used locus-specific fixed sequence oligos thatwere complementary to adjacent regions on the nucleic acid of interest,and thus no gap was created by hybridization of these oligos. In thisformat, no polymerase was required, and a ligase was used to seal thesingle nick between the oligos.

A sixth exemplary format used allele-specific fixed sequence oligos andlocus-specific bridging oligos, where there was a short base gap of fivebases between the loci region complementary to the fixed sequenceoligos. The locus-specific bridging oligo in this example was a 5mercomplementary to the regions directly adjacent to the regionscomplementary to the first and second fixed sequence oligos. In thisformat, no polymerase was required, and a ligase was used to seal thetwo nicks between the oligos.

A seventh exemplary format used locus-specific fixed sequence oligos anda locus-specific bridging oligo, where there was a shorter base gap offive bases containing a SNP in the region complementary to the bridgingoligo. Allele-specific bridging oligos corresponding to the possibleSNPs were included in the hybridization and ligation reaction. In thisformat, no polymerase was required, and a ligase was used to seal thetwo nicks between the oligos. SNP base discrimination in this assayformat derived from hybridization specificity and the tendency of theligase to not seal nicks adjacent to mismatched bases.

An eighth exemplary format used locus-specific fixed sequence oligos andtwo adjacent locus-specific bridging oligos, where there was a 10 basegap between the regions complementary to the first and second fixedsequence oligos. Locus-specific bridging oligos were included in theligation reaction, with the gap requiring two contiguous 5mers to bridgethe gap. In this format, no polymerase was required, and a ligase wasused to seal the three nicks between the oligos.

For each of the above-described assay formats, an equimolar pool (40 nMeach) of sets of first and second loci- or allele-specific fixedsequence oligonucleotides was created from the oligos prepared as setforth in Example 2. A separate equimolar pool (20 μM each) of bridgingoligonucleotides was likewise created for the assay processes based onthe sequences of the selected genomic loci.

100 μg of strepavidin beads were transferred into the wells of a 96 wellplate, and the supernatant was removed. 60 μl BB2 buffer (100 mM Tris pH8.0, 10 mM EDTA, 500 mM NaCl₂, 58% formamide, 0.17% Tween-80), 10 μL 40nM fixed sequence oligo pool and 30 μL of the biotinylated template DNAprepared in Example 2 were added to the beads. The plate was sealed withan adhesive plate sealer and vortexed at 3000 rpm until beads wereresuspended. The oligos were annealed to the template DNA by incubationat 70° C. for 5 minutes, followed by slow cooling to room temperature.

The plate was placed on a raised bar magnetic plate for 2 minutes topull the magnetic beads and associated DNA to the side of the wells. Thesupernatant was removed by pipetting, and was replaced with 50 μl of 60%BB2 (v/v in water). The beads were resuspended by vortexing, placed onthe magnet again, and the supernatant was removed. This bead washprocedure was repeated once using 50 μl 60% BB2, and repeated twice moreusing 50 μl wash buffer (10 mM Tris pH 8.0, 1 mM EDTA, 50 mM NaCl₂).

The beads were resuspended in 37 μl ligation reaction mix consisting of1× Taq ligase buffer (Enzymatics, Beverly, Mass.), 1U Taq ligase, and 2μM bridging oligo pool (depending on the assay format), and incubated at37° C. for one hour. Where appropriate, and depending on the assayformat, a non-proofreading thermostable polymerase plus 200 nM each dNTPwas included in this mixture. The plate was placed on a raised barmagnetic plate for 2 minutes to pull the magnetic beads and associatedDNA to the side of the wells. The supernatant was removed by pipetting,and was replaced with 50 μL wash buffer. The beads were resuspended byvortexing, placed on the magnet again, and the supernatant was removed.The wash procedure was repeated once.

To elute the products from the strepavidin beads, 30 μl of 10 mM Tris 1mM EDTA, pH 8.0 was added to each well of 96-well plate. The plate wassealed and mixed using an IKA vortexer for 2 minutes at 3000 rpm toresuspend the beads. The plate was incubated at 95° C. for 1 minute, andthe supernatant aspirated using an 8-channel pipetter. 25 μl ofsupernatant from each well was transferred into a fresh 96-well platefor universal amplification.

Example 4: Universal Amplification of Tandem Ligated Products

The polymerized and/or ligated nucleic acids were amplified usinguniversal PCR primers complementary to the universal sequences presentin the first and second fixed sequence oligos hybridized to the loci ofinterest. 25 μl of each of the reaction mixtures of Example 3 were usedin each amplification reaction. A 50 μl universal PCR reactionconsisting of 25 μl eluted ligation product plus 1× Pfusion buffer, 1MBetaine, 400 nM each dNTP, 1 U Pfusion error-correcting thermostable DNApolymerase (Thermo Fisher, Waltham Mass.), and the following primerpairs: TAATGATACGGCGACCACCGAGATCTACACCGGCGTTATGCGTCGAGA (SEQ ID NO:3)and TCAAGCAGAAGACGGCATACGAGATXAAACGACGCGATCATCGGTCCCC GCAA (SEQ IDNO:4), where X represents one of 96 different sample indices used touniquely identify individual samples prior to pooling and sequencing.The PCR was carried out under stringent conditions using a BioRadTetrad™ thermocycler.

10 μl of universal PCR product from each of the samples were pooled andthe pooled PCR product was purified using AMPureXP™ SPRI beads(Beckman-Coulter, Danvers, Mass.), and quantified using Quant-iT™PicoGreen, (Invitrogen, Carlsbad, Calif.).

Example 5: Detection and Analysis of Selected Loci

The purified PCR products of each assay format were sequenced on asingle lane of a slide on an Illumina HiSeq™ 2000 (Illumina, San Diego,Calif.). Sequencing runs typically give rise to ˜100M raw reads, ofwhich ˜85M (85%) mapped to expected assay structures. This translated toan average of ˜885K reads/sample across the experiment, and (in the caseof an experiment using 96 loci) 9.2K reads/replicate/locus across 96loci. The mapped reads were parsed into replicate/locus/allele counts,and various metrics were computed for each condition, including:

Yield: a metric of the proportion of input DNA that was queried insequencing, computed as the average number of unique reads per locus(only counting unique identification index reads per replicate/locus)divided by the total number of genomic equivalents contained in theinput DNA.

80 percentile locus frequency range: a metric of the locus frequencyvariability in the sequencing data, interpreted as the fold range thatencompasses 80% of the loci. It was computed on the distribution oftotal reads per locus, across all loci, as the 90^(th) percentile oftotal reads per locus divided by the 10^(th) percentile of the totalreads per locus.

SNP error rate: a metric of the error rate at the SNP position, andcomputed as the proportion of reads containing a discordant base at theSNP position.

These results are summarized in Table 1:

TABLE 1 Results Summary of Tandem Ligation Assay Formats FIXED 80%SEQUENCE BRIDGING LOC SNP ASSAY OLIGO (1^(st) and/or OLIGO ENZYME FREQERROR FORMAT 2^(nd)) USED USED YIELD RANGE RATE 1 LOCUS-SPECIFIC Locuspol + lig 9.5% 5.3 0.18% specific 2 LOCUS-SPECIFIC No pol + lig 1.4%58.3 0.19% 3 ALLELE- No pol + lig 0.4% 61.7 1.00% SPECIFIC 4 ALLELE-Locus Taq lig 5.0% 5.9 0.92% SPECIFIC specific 4 ALLELE- Locus T4 lig5.3% 4.4 0.95% SPECIFIC specific 5 LOCUS-SPECIFIC No Taq lig 22.5% 1.7N/A 6 LOCUS-SPECIFIC Locus Taq lig 12.5 2.9 N/A specific 7LOCUS-SPECIFIC Allele Taq lig 14.3 2.8 0.20% specific 8 LOCUS-SPECIFIC 2Locus Taq lig 18.5% 2.8 N/A specific

Table 1 indicates that the locus-specific tandem ligation assay using abridging oligo converted template DNA into targeted product with highyield (˜10%), with a high proportion of product derived from targetedloci (15% of reads did not contain expected assay structures), withlimited locus bias (80% of loci fall within a ˜5-fold concentrationrange), and with high SNP accuracy (0.2% SNP error rate). Thelocus-specific tandem ligation assay without the use of a bridging oligoproduced reduced yields and substantial locus bias, but still producedhigh accuracy SNP genotyping data. The allele-specific tandem ligationassay with a bridging oligo produced intermediate yields compared to thelocus-specific assay using both T4 and Taq ligase, but still producedlimited locus bias and high accuracy SNP genotyping data. Theallele-specific tandem ligation assay without a bridging producedreduced yields and substantial locus bias, but still produced highaccuracy SNP genotyping data.

Assay formats six through eight showed that template DNA can beconverted into targeted product with high yield (12-18%), with a highproportion of product derived from targeted loci (˜76% of readscontained expected assay structures), and with limited locus bias (80%of loci fall within a 2-3-fold concentration range). FIG. 5 illustratesthe genotyping performance that was obtained using assay format seven,comparing the sequence counts for the two alleles of all polymorphicassays observed in a single sample. Note the clear separation of thehomozygous and heterozygous clusters, as well as the low backgroundcounts observed amongst the homozygous clusters.

Example 6: Detection of Aneuploidy in Patient Samples from PregnantSubjects

The assay systems of the invention were used in the detection ofpolymorphisms and chromosomal abnormalities in two separate cohorts ofpregnant females. A first cohort of 190 normal, 36 T21, and 8 T18pregnancies and a second cohort of 126 normal, 36 T21, and 8 T18pregnancies were tested for fetal aneuploidy. The chromosomalaneuploidies were detected using 576 chromosome 21 and 576 chromosome 18assays, pooled together and assayed in a single reaction, as set forthbelow.

The elements used in the aneuploidy detection assays are illustrated inFIG. 6. The cfDNA 601 isolated from maternal samples was used as atemplate for hybridization, ligation, and amplification of multipleselected loci from both chromosome 21 and chromosome 18 in each maternalsample. Three oligonucleotides were hybridized to each selected locus tocreate ligation products for amplification and detection. The left (orfirst) fixed sequence oligonucleotide comprised a region complementaryto a selected locus 609 and a first universal primer region 611. Theright (or second) fixed sequence oligonucleotide 605 comprised a secondregion complementary to the selected locus 613 and a second universalprimer region 615. The bridging oligonucleotides 607 used were designedso that each would hybridize to bridging regions of two or more selectedloci used in the aneuploidy detection assay. When the fixed sequenceoligonucleotides 603, 605 and the bridging oligonucleotide 607hybridized to the complementary region on the cfDNA 601, their terminiformed two nicks. Upon ligation of the hybridized oligonucleotides tothe cfDNA, a ligation product was created for each selected locuscomprising 603, 605 and 607 which was used as a template foramplification primers 619, 621.

Two amplification primers 619, 621 comprising regions complementary tothe first and second universal primer regions, respectively, were thenused to amplify the ligation product. This amplification productcomprised the sequence of the selected locus. The right amplificationprimer also comprised a sample index 617 to identify the particularsample from which the locus was obtained in the multiplexed assay.Amplification with 96 distinct right amplification primers 629 enabledpooling and simultaneous sequencing of 96 different amplificationproducts on a single lane.

The amplification primers 619, 621 also contained a left clustersequence 623 (TAATGATACGGCGACCACCGA)(SEQ ID NO:7) and a right clustersequence 625 (ATCTCGTATGCCGTCTTCTGCTTGA)(SEQ ID NO:8) that supportedcluster amplification for sequencing using the Illumina HiSeq™ 2000system (Illumina, San Diego, Calif.). A sequencing primer 627 comprisingthe first universal primer sequence was used to determine the sequenceof the amplification product, and a second sequencing primer 629 wasused to determine the sample index 617 of the amplification product.

Briefly, approximately 10 mL peripheral blood was collected from eachpatient into a BCT tube (Streck, Omaha, Nebr.), which was shipped viaovernight courier to Tandem Diagnostics. Plasma was isolated from BCTtubes within 72 h of blood collection by centrifugation at 1600 g for 10m. The plasma was transferred to a second tube and centrifuged at 16000g for 10 m to remove any remaining cells. cfDNA was isolated from 4-5 mLplasma per patient. Approximately 15 ng cfDNA was isolated from eachpatient sample and arrayed into individual wells of a 96 well plate. Allsubsequent processing occurred on multiplexed batches of up to 96 cfDNApatient samples per array system method.

cfDNA isolated from the maternal samples in each well was biotinylatedprecipitated and resuspended in 30 uL TE as in Example 3 above. Thebiotinylated template DNA was mixed with 100 ug MyOneC1streptavidin-coated magnetic beads (Life Technologies, Carlsbad,Calif.), 60 μl BB2 buffer (100 mM Tris pH 8.0, 10 mM EDTA, 500 mM NaCl₂,58% formamide, 0.17% Tween-80), and 10 μL of pooled 40 nM left 603 andright 605 fixed sequence oligonucleotides.. The mixture was heated to70° C., and cooled 2 hours. The beads were then magnetically immobilizedto the side of the well, washed twice with 50 uL 60% BB2 (v/v with H2O),washed twice more with 50 μl wash buffer (10 mM Tris pH 8.0, 1 mM EDTA,50 mM NaCl2), and then resuspended in a 50 μL reaction containing 1U Taqligase (Enzymatics, Beverly Mass.), 1× Taq ligase buffer (Enzymatics),and 10 uM of a 5′-phosphorylated 5mer bridging oligonucleotide 607. Themixture was incubated at 37° C. for 1 hour. The beads were againmagnetically immobilized to the side of the well, washed twice with 50uL wash buffer and then resuspended in 30 μL TE.

The ligation products were eluted from the immobilized beads byincubation at 95° C. for 3 minutes. The eluted ligation products wereamplified by 26 cycles of PCR in a 50 uL reaction containing 1U Pfusion(Finnzymes), 1M Betaine, 1× Pfusion buffer, and 400 nM left and rightamplification primers (619, 621 respectively). The right primercontained a 7 base sample index (617) that enabled 96 sample multiplexedsequencing on the HiSeq2000 (Illumina, San Diego, Calif.). The sequenceof the left fixed sequence oligo was:

(SEQ ID NO: 5) TAATGATACGGCGACCACCGAGATCTACACCGGCGTTATGCGTCGAGAC

And the sequence of the right fixed sequence oligo was:

(SEQ ID NO: 6) TCAAGCAGAAGACGGCATACGAGATNNNNNNNAAACGACGCGATCATCGGTCCCCGCAAT

Amplification products from a single 96 well plate were pooled in equalvolume, and the pooled amplification products were purified withAMPureXP™ SPRI beads (Beckman-Coulter, Danvers, Mass.) according to themanufacturer's instructions. Each purified pooled library was used astemplate for cluster amplification on an Illumina TruSeq v2 SR clusterkit flow cell (Illumina, San Diego, Calif.) according to manufacturer'sprotocols. The slide was processed on an Illumina HiSeq™ 2000 (Illumina,San Diego, Calif.) to produce 56 bases of locus-specific sequence from aleft sequence primer 623 and a separate read of 8 bases of samplespecific sequence was obtained from the second sequence primer 625. Anaverage of 903K raw reads per sample were collected. An average of 876K(97%) of the reads was assigned to expected assay structures.

FIG. 7 shows exemplary data for a subset of the patient samples from thesecond cohort, which were all analyzed in one multiplexed assay on asingle lane of a sequencing run. Initially 96 different samples were runin this particular run, but—six samples were later excluded from thisanalytical set as not meeting sample quality control thresholds.

A trimmed mean was calculated for each chromosome 18 and chromosome 21for the samples based on reads produced in the assay. The trimmed meanwas computed by removing 10% of high and low counts for each chromosomeby sample. The detected amplification products corresponding to thevarious selected loci were used to compute a chromosome 21 proportionmetric and a chromosome 18 proportion metric for each sample. Forchromosome 21 proportion, this was calculated as the trimmed mean ofcounts in the 384 chromosome 21 selected loci divided by the sum oftrimmed means of counts for all 576 chromosome 21 loci and 576chromosome 18 loci for each sample.

On average 834 read counts were observed per selected locus in thematernal samples of the first cohort, and 664 read counts were observedper selected locus from the second cohort. These counts were used tocompute chromosome proportion z-scores for chromosome 21 and chromosome18.

Briefly, the z-scores were calculated by scaling the median per locuscount to a common value (e.g., 1000) for each sample, and the scaledcounts were transformed by log base 2. An RMA log linear modeling andmedian polish were performed (Bolstad, B. M et al. (2003) Bioinformatics19(2): 185-193; Rafael. A. (2003) Nucleic Acids Research 31(4):e15;Irizarry, R A et al. (2003) Biostatistics 4(2):249-64) to estimatechromosome effects, locus effects, sample effects, and residuals. Theestimated chromosome effects were set to a common value, e.g., 0, and2^(chromosome effect+sample effect+residual) was calculated for eachlocus to create normalized counts. The Z scores were scaled usingiterative censoring so that they had a mean of 0 and a standarddeviation of 1.

Data obtained from the first cohort of samples was used to determinefirst cohort z-scores for chromosome 21 and chromosome 18 areillustrated in FIGS. 8 and 9, respectively. The normal samples are shownas dark grey diamonds, and the samples with a trisomy are shown as lightgrey diamonds. 179/180 (99.4%) normal samples (dark grey diamonds) hadz-scores <3; one normal sample had a chromosome 21 z-score of 3.4 and achromosome 18 z-score of 3.0. 35/35 (100%) T21 and 7/7 (100%) T18samples had chromosome proportion z-scores >3. The mean T18 z-score was8.5, and the range was 5.8-10.9. The mean T21 z-score was 11.5, and therange was 6.1-19.8.

The data provided in FIG. 7 was combined with data from the remainingsamples of the second cohort to determine z-scores for chromosome 21 andchromosome 18 are illustrated in FIGS. 10 and 11, respectively. Thenormal samples are shown as dark grey diamonds, and the samples with atrisomy are shown as light grey diamonds. 125/125 normal samples hadz-scores <3, 36/36 (100%) T21 and 8/8 (100%) T18 samples hadz-scores >3. The mean T18 z-score was 9.5 and the range was 5.1-19.8.The mean T21 z-score was 11.4 and the range was 3.4-21.8.

In addition to the detection of aneuploidy in these cohorts, specificpolymorphisms were also determined for these samples in a same assay.Specific information was obtained for individual loci as well as moregeneral polymorphic information, such as the number of loci in which thefetal locus displayed a single nucleotide polymorphism in one alleledifferent from the single nucleotide polymorphisms at the maternal locus(FIG. 7, #Locus DiffPoly). This determination also identified thepresence of specific polymorphisms in the fetal genome. For example, thestatus of three exemplary polymorphism were determined using acombination of bridging oligos that were designed to bind to both the Aand the T residue in the following exemplary polymorphic regions:

TABLE 2 Individual Polymorphisms Queried Using the Invention AssayChromosome Location RSID Ch01 01_010303942 rs11582123TTTACATGTCTTTGGGCATTTTAGGT[A/T]GAGTGAAATCTAGGCCTTG CAAATC (SEQ ID NO: 7)Ch03 03_098690592 rs2470750TTGTGTAACGTTAACCTCAGGGACCA[A/T]GAGATGTACTTAGTATTAA TTTGCC (SEQ ID NO: 8)Ch04 04_055495793 rs6815910GGAAGAAGTGCAGTGTAGTAGACAAC[A/T]CTGGCATTGTGTTTTGTGA ACTGGG (SEQ ID NO: 9)

TABLE 3 Predicted Maternal and Fetal Status for SNP rs11582123.Predicted A T Predicted Maternal Sample SNP counts counts Fetal StatusStatus 1 rs11582123 294 26 A/T A/A 2 181 134 A/A A/T 4 34 330 A/T T/T 5241 21 A/T A/A 6 166 134 A/T A/T 7 137 182 T/T A/T 8 199 135 A/A A/T 9 0267 T/T T/T 10 0 284 T/T T/T 11 151 154 A/T A/T 12 294 1 A/T A/A 13 131114 A/A A/T 14 118 159 T/T A/T 15 257 10 A/T A/A 16 309 31 A/T A/A 17 20289 A/T T/A 18 137 166 T/T A/T 19 138 143 A/T A/T 20 24 242 A/T T/T 21140 161 A/T A/T 22 159 118 A/A A/T 23 119 122 A/T A/T 24 0 250 T/T T/T25 0 285 T/T T/T 26 120 130 A/T A/T 28 134 113 A/A A/T 29 109 118 A/TA/T 30 0 271 T/T T/T 31 148 139 A/T A/T 32 29 253 A/T T/T 33 0 304 T/TT/T 34 0 278 T/T T/T 35 103 188 T/T A/T 36 18 269 A/T T/T 37 279 34 A/TA/A 38 0 250 T/T T/T 39 0 263 T/T T/T 40 136 142 A/T A/T 41 147 145 A/TA/T 42 15 270 A/T T/T 43 44 222 A/T T/T 44 140 159 T/T A/T 45 0 259 T/TT/T 46 1 304 T/T T/T 47 162 127 A/A A/T 48 0 335 T/T T/T 49 1 247 T/TT/T 50 153 154 A/T A/T 51 118 182 T/T A/T 52 145 134 A/T A/T 53 146 132A/T A/T 54 7 319 A/T T/T 55 152 174 T/T A/T 56 1 319 T/T T/T 57 147 150A/T A/T 58 136 157 T/T A/T 59 83 162 A/A T/T 60 14 215 A/T T/T 61 157121 A/A A/T 62 281 0 A/A A/A 63 0 260 T/T T/T 64 0 305 T/T T/T 65 18 252A/T T/T 66 0 303 T/T T/T 67 99 161 T/T A/T 68 141 127 A/T A/T 69 0 237T/T T/T 70 0 315 T/T T/T 71 132 139 A/T A/T 73 112 120 A/T A/T 75 1 268T/T T/T 76 166 123 A/A A/T 78 0 245 T/T T/T 79 12 264 A/T T/T 80 15 281A/T T/T 81 21 269 A/T T/T 82 108 160 T/T A/T 83 106 144 T/T A/T 84 137135 A/T A/T 85 115 151 T/T A/T 86 0 262 T/T T/T 87 0 269 T/T T/T 89 0284 T/T T/T 90 0 261 T/T T/T 91 143 137 A/T A/T 92 0 308 T/T T/T 93 1256 T/T T/T 94 158 105 A/A A/T 95 149 103 A/A A/T

TABLE 4 Predicted Maternal and Fetal Status for SNP rs2470750. PredictedA T Predicted Maternal Sample SNP counts counts Fetal Status Status 1rs2470750 243 15 A/T A/A 2 265 0 A/A A/A 4 170 107 A/A A/T 5 196 30 A/TA/A 6 141 144 A/T A/T 7 139 137 A/T A/T 8 272 0 A/A A/A 9 218 0 A/A A/A10 216 0 A/A A/A 11 228 0 A/A A/A 12 6 224 A/T T/T 13 126 93 A/A A/T 14125 123 A/T A/T 15 234 20 A/T A/A 16 147 113 A/A A/T 17 235 2 A/T A/A 18129 142 A/T A/T 19 132 114 A/A A/T 20 214 0 A/A A/A 21 1 245 T/T T/T 22141 111 A/A A/T 23 135 128 A/A A/T 24 121 160 T/T A/T 25 209 21 A/T A/A26 0 239 T/T T/T 27 203 4 A/T A/A 28 101 115 T/T A/T 29 212 10 A/T A/A30 86 101 T/T A/T 31 118 116 A/T A/T 32 135 121 A/A A/T 33 111 128 T/TA/T 34 120 118 A/T A/T 35 246 0 A/A A/A 36 113 115 A/T A/T 37 96 126 T/TA/T 38 107 88 A/A A/T 39 241 0 A/A A/A 40 116 118 A/T A/T 41 135 89 A/AA/T 42 129 85 A/A A/T 43 0 205 T/T T/T 44 138 88 A/A A/T 45 129 86 A/AA/T 46 108 123 T/T A/T 47 14 246 A/T T/T 48 129 148 T/T A/T 49 108 110A/T A/T 50 120 124 A/T A/T 51 212 22 A/T A/T 52 237 0 A/A A/A 53 104 147T/T A/T 54 134 126 A/T A/T 55 128 82 A/A A/T 56 225 5 A/T A/A 57 213 11A/T A/A 58 125 116 A/T A/T 59 226 1 A/A A/A 60 103 119 T/T A/T 61 84 91T/T A/T 62 130 104 A/A A/T 63 251 0 A/A A/A 64 243 0 A/A A/A 65 127 115A/A A/T 66 113 104 A/A A/T 67 26 190 A/T T/T 68 80 83 A/T A/T 69 122 132T/T A/T 70 0 235 T/T T/T 71 90 123 T/T A/T 73 174 0 A/A A/A 75 0 233 T/TT/T 76 220 0 A/A A/A 78 115 115 A/T A/T 79 112 144 T/T A/T 80 10 248 A/TT/T 81 241 0 A/A A/A 82 228 0 A/A A/A 83 243 16 A/T A/A 84 133 104 A/AA/T 85 101 99 A/T A/T 86 1 209 A/A A/A 87 224 7 A/T A/A 89 122 101 A/AA/T 90 130 89 A/A A/T 91 128 151 T/T A/T 92 231 0 A/A A/A 93 107 118 T/TA/T 94 93 100 A/T A/T 95 132 119 A/A A/T

TABLE 5 Predicted Maternal and Fetal Status for SNP rs6S15910. PredictedA T Predicted Maternal Sample SNP counts counts Fetal Status Status 1rs6815910 295 32 A/T A/A 10 133 107 A/A A/T 11 115 131 T/T A/T 12 311 10A/T A/A 13 18 252 A/T T/T 14 132 178 T/T A/T 15 288 0 A/A A/A 16 325 1A/A A/A 17 11 276 A/T T/T 18 282 0 A/A A/A 19 131 133 A/T A/T 2 7 311A/T T/T 20 135 116 A/A A/T 21 121 140 T/T A/T 22 287 11 A/T A/A 23 148146 A/T A/T 24 185 138 A/A A/T 25 116 126 T/T A/T 26 235 0 A/A A/A 27288 0 A/A A/A 28 242 0 A/A A/A 29 239 12 A/T A/A 30 235 24 A/T A/A 31126 148 T/T A/T 32 25 256 A/T T/T 33 286 1 A/A A/A 34 158 156 A/T A/T 35287 0 A/A A/A 36 118 133 T/T A/T 37 163 119 A/A A/T 38 273 10 A/T A/A 39132 148 T/T A/T 4 0 343 T/T T/T 40 143 177 T/T A/T 41 0 308 T/T T/T 42297 0 A/A A/A 43 117 130 T/T A/T 44 296 1 A/A A/A 45 276 0 A/A A/A 46140 134 A/T A/T 47 158 139 A/A A/T 48 0 304 T/T T/T 49 251 13 A/T A/A 5138 115 A/A A/T 50 142 162 T/T A/T 51 0 306 T/T T/T 52 249 21 A/T A/A 53111 170 T/T A/T 54 140 151 A/T A/T 55 102 217 T/T A/T 56 315 0 A/A A/A57 123 158 T/T A/T 58 146 168 T/T A/T 59 226 50 A/T A/A 6 309 0 A/A A/A60 122 133 T/T A/T 61 240 28 A/T A/A 62 132 124 A/T A/T 63 291 9 A/T A/A64 0 304 T/T T/T 65 273 0 A/A A/A 66 154 139 A/T A/T 67 145 153 A/T A/T68 110 163 T/T A/T 69 131 134 A/T A/T 7 186 127 A/A A/T 70 167 163 A/TA/T 71 238 26 A/T A/A 73 18 244 A/T T/T 75 130 129 A/T A/T 76 133 113A/A A/T 78 237 2 A/T A/A 79 0 278 T/T T/T 8 192 159 A/A A/T 80 153 131A/A A/T 81 25 229 A/T T/T 82 0 256 T/T T/T 83 152 142 A/T A/T 84 290 2A/T A/A 85 270 0 A/A A/A 86 0 242 T/T T/T 87 150 134 A/A A/T 89 169 117A/A A/T 9 271 1 A/A A/A 90 109 144 T/T A/T 91 261 12 A/T A/A 92 258 0A/A A/A 93 0 309 T/T T/T 94 116 146 T/T A/T 95 123 116 A/T A/T

The location of these SNPS is denoted using dbSNP version 132 andGRCH37/UCSC hg 19. The data for these polymorphisms was obtained in thesame data set as the aneuploidy data illustrated in FIGS. 10 and 11.Thus, a single assay demonstrated the ability to identify fetalaneuploidy, polymorphic differences between fetal and maternal loci, andthe actual SNP information for selected fetal loci in a single assay.

Numerous variations may be made by persons skilled in the art withoutdeparture from the spirit of the invention. The scope of the inventionwill be measured by the appended claims and their equivalents. Theabstract and the title are not to be construed as limiting the scope ofthe present invention, as their purpose is to enable the appropriateauthorities, as well as the general public, to quickly determine thegeneral nature of the invention. In the claims that follow, unless theterm “means” is used, none of the features or elements recited thereinshould be construed as means-plus-function limitations pursuant to 35U.S.C. § 112, ¶6.

What is claimed is:
 1. A method of detecting a presence or absence ofcopy number variation (CNV) in a mixed sample from a single subject,said mixed sample comprising nucleic acids from at least two sourcescomprising a major source and a minor source, the method comprising: a)hybridizing at least 24 first sets of two fixed sequenceoligonucleotides to the nucleic acids in the mixed sample, wherein eachfirst set of two fixed sequence oligonucleotides is complementary to alocus in a first region of interest, each first set of two fixedsequence oligonucleotides comprising universal primer regions, andwherein melting temperatures (T_(m)s) of first fixed sequenceoligonucleotides of each first set of two fixed sequenceoligonucleotides vary in a range of two degrees centigrade; b)hybridizing at least 24 second sets of two fixed sequenceoligonucleotides to the nucleic acids in the mixed sample, wherein eachsecond set of two fixed sequence oligonucleotides is complementary to alocus in a second region of interest, each second set of two fixedsequence oligonucleotides comprising universal primer regions, andwherein T_(m)s of first fixed sequence oligonucleotides of each secondset of two fixed sequence oligonucleotides vary in a range of twodegrees centigrade; c) extending one of said hybridized fixed sequenceoligonucleotides of each first set of two fixed sequenceoligonucleotides with a polymerase between the hybridizedoligonucleotides of each first set of two fixed sequenceoligonucleotides to produce adjacently hybridized fixed sequenceoligonucleotides and extending one of said hybridized fixed sequenceoligonucleotides of each second set of two fixed sequenceoligonucleotides with a polymerase between the hybridizedoligonucleotides of each second set of two fixed sequenceoligonucleotides to produce adjacently hybridized fixed sequenceoligonucleotides; d) ligating the adjacently hybridized fixed sequenceoligonucleotides of each first set of two fixed sequenceoligonucleotides to create contiguous ligation products and ligating theadjacently hybridized fixed sequence oligonucleotides of each second setof two fixed sequence oligonucleotides to create contiguous ligationproducts; e) amplifying the contiguous ligation products using theuniversal primer regions on each first and second sets of two fixedsequence oligonucleotides to produce amplification products; f)detecting the amplification products; and g) detecting a copy numbervariation of the first region of interest relative to the second regionof interest.
 2. The method of claim 1, wherein the subject comprises apregnant female such that the nucleic acids of the major source comprisematernal nucleic acids and the nucleic acids of the minor sourcecomprise fetal nucleic acids.
 3. The method of claim 1, wherein thesubject comprises an individual having normal cells and cancerous cellssuch that the nucleic acids of the major source comprise nucleic acidsof the normal cells and the nucleic acids of the minor source comprisenucleic acids of the cancerous cells.
 4. The method of claim 1, whereinthe subject comprises a transplant patient such that the nucleic acidsof the major source comprise nucleic acids of the patient and thenucleic acids of the minor source comprise nucleic acids of a donororgan, and wherein said copy number variation is detected in said majorsource or said minor source.
 5. The method of claim 1, wherein thesubject comprises an individual infected with an organism such that thenucleic acids of the major source comprise nucleic acids of theindividual and the nucleic acids of the minor source comprise nucleicacids of the infectious organism, and wherein said copy number variationis detected in said major source or said minor source.
 6. The method ofclaim 1, wherein each locus in the second region of interest comprisesone or more polymorphisms.
 7. The method of claim 1, wherein the firstregion of interest and the second region of interest are on differentchromosomes.
 8. The method of claim 1, wherein at least one locus doesnot comprise a polymorphism.
 9. The method of claim 1, wherein one orboth sets of the fixed sequence oligonucleotides comprises at least oneindex.
 10. The method of claim 9, wherein the amplification products aredetected by sequencing the at least one index.
 11. The method of claim9, wherein the at least one index comprises a locus index.
 12. Themethod of claim 9, wherein the at least one index comprises a sampleindex.
 13. The method of claim 1, wherein the amplification products aredetected by high throughput sequencing.
 14. The method of claim 1,wherein the amplification products are detected on an array.
 15. Themethod of claim 1, wherein one or both sets of fixed sequenceoligonucleotides comprises an oligonucleotide 5′ of each locus, anoligonucleotide 3′ of each locus, and a bridging oligonucleotide. 16.The method of claim 1, wherein one or more bridging oligonucleotides isadded prior to the step of extension.
 17. The method of claim 1, whereinthe amplification products are isolated as individual molecules prior todetection.
 18. The method of claim 17, wherein the individual isolatedamplification products are further amplified to create identical copiesof all or a portion of the individual amplification products prior todetection.
 19. A method of detecting a presence or absence of geneticvariation in a mixed sample from a single subject, said mixed samplecomprising nucleic acids from at least two sources comprising a majorsource and a minor source, the method comprising: a) hybridizing atleast 24 sets of two fixed sequence oligonucleotides to the nucleicacids in the mixed sample, wherein each set of two fixed sequenceoligonucleotides is complementary to a polymorphic locus in a region ofinterest, each set of two fixed sequence oligonucleotides comprisinguniversal primer regions, and each locus in the region of interesthaving different genotypes in the major source and the minor source, andwherein T_(m)s of first fixed sequence oligonucleotides of each set oftwo fixed sequence oligonucleotides vary in a range of two degreescentigrade; b) extending one of said hybridized two fixed sequenceoligonucleotides of each set with a polymerase between the hybridizedoligonucleotides of the set to produce adjacently hybridized fixedsequence oligonucleotides; c) ligating the adjacently hybridizedoligonucleotides to create contiguous ligation products complementary tothe locus; d) amplifying the contiguous ligation products using theuniversal primer regions on the two fixed sequence oligonucleotides ofeach set of fixed sequence oligonucleotides to produce amplificationproducts; e) detecting the amplification products; and f) detecting apresence or absence of genetic variation in the locus of interestbetween the major and minor sources from the detected amplifiedproducts.